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RegulationoftumorigenicWntsignalingbycyclooxygenase-2,5-lipoxygenaseandtheirpharmacological...

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Pharmacology & Therapeutics 157 (2016) 43–64

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Regulation of tumorigenic Wnt signaling bycyclooxygenase-2, 5-lipoxygenase and their pharmacologicalinhibitors: A basis for novel drugs targeting cancer cells?

Jessica Roos a,⁎,1, Sabine Grösch b,1, Oliver Werz c,1, Peter Schröder d,1, Slava Ziegler d, Simone Fulda e,Patrick Paulus f, Anja Urbschat g, Benjamin Kühn a, Isabelle Maucher a, Jasmin Fettel a, Thomas Vorup-Jensen h,Matthias Piesche h, Carmela Matrone h, Dieter Steinhilber a, Michael J. Parnham i,1, Thorsten J. Maier a,h,⁎⁎,1a Institute of Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germanyb Pharmazentrum Frankfurt/ZAFES, Institute of Clinical Pharmacology, Goethe-University, 60590, Frankfurt am Main, Germanyc Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University of Jena, Philosophenweg 14, 07743 Jena, Germanyd Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germanye Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germanyf Department of Anesthesiology and Operative Intensive Care Medicine, Kepler University Hospital, 4021 Linz, Austriag Department of Urology and Pediatric Urology, Philipps-University Hospital, 35043 Marburg, Germanyh Aarhus University, Department of Biomedicine & Center for Study and Prevention of Neurodegenerative Inflammation (NEURODIN), Bartholins Allé 6, 8000 Aarhus C, Denmarki Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group for Translational Medicine and Pharmacology TMP, 60596 Frankfurt am Main, Germany

Abbreviations: AML, acute myeloid leukemia; APC, admyeloid leukemia; COX, cyclooxygenase; CSC, cancer stemstem cell; LEF, lymphoid enhancer factor; LSC, leukemic santi-inflammatory drugs; NF-κB, nuclear factor-κB; PG, pTCF, T-cell factor.⁎ Correspondence to: J. Roos, Institute of Pharmaceutic⁎⁎ Correspondence to: T.J. Maier, Aarhus University, Dep

E-mail addresses: roos@pharmchem.uni-frankfurt.de (1 These authors contributed equally.

http://dx.doi.org/10.1016/j.pharmthera.2015.11.0010163-7258/© 2015 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Available online 6 November 2015

Keywords:Wnt inhibitorTumor cellsLeukemic stem cellsLeukotrienesNon-steroidal anti-inflammatory drugs

Canonical Wnt signaling is a highly conserved pathway with a prominent role in embryogenic development,adult tissue homeostasis, cell polarization, stem cell biology, cell differentiation, and proliferation. Furthermore,canonical Wnt signaling is of pivotal importance in the pathogenesis of a number of cancer types and cruciallyaffects tumor initiation, cancer cell proliferation, cancer cell apoptosis, and metastasis.Reports over the last decade have provided strong evidence for a pathophysiological role ofWnt signaling in non-malignant classical inflammatory and neurodegenerative diseases.Although, several agents suppressing theWnt pathway at different levels have been identified, the developmentof clinically relevant Wnt-inhibiting agents remains challenging due to selectivity and toxicity issues.Several studies have shown that long-term administration of non-steroidal anti-inflammatory drugs protectsagainst colon cancer and potentially other tumor types by interfering both with the COX and the Wnt pathway.Our own studies have shown that non-steroidal anti-inflammatory drugs suppress Wnt signaling by targetingthe pro-inflammatory enzyme5-lipoxygenasewhich is the key enzymepathophysiologically involved in the syn-thesis of leukotrienes.Furthermore, we found a direct link between the 5-lipoxygenase andWnt signaling pathways, which is essentialfor the maintenance of leukemic stem cells. Accordingly, genetic and pharmacological inhibition of 5-lipoxygenase led to an impairment of Wnt-dependent acute and chronic myeloid leukemic stem cells.We believe that 5-lipoxygenase inhibitors might represent a novel type of Wnt inhibitor activating a potentiallynaturally occurring novel mechanism of suppression of Wnt signaling that is non-toxic, at least in mice, and ispotentially well tolerated in patients.

© 2015 Elsevier Inc. All rights reserved.

enomatous polyposis coli; CBP, CREB-binding protein; CK1, casein kinase 1; CLL, chronic lymphatic leukemia; CML, chroniccell; Dvl, disheveled; FLAP, 5-LO-activating protein; Fzd, Frizzled; GSK3β, glycogen synthase kinase 3β; HSC, hematopoietic

tem cell; 5-LO, 5-lipoxygenase; LRP, low density lipoprotein receptor-related protein; LT, leukotriene; NSAIDs, non-steroidalrostaglandin; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PPAR, peroxisome proliferator activated receptor;

al Chemistry/ZAFES, Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt/Main, Germany. Tel.: +49 69 7982 9341.artment of Biomedicine, Bartholins Allé 6, 8000 Aarhus C, Denmark. Tel.: +45 87167681; fax: +49 69 798 29323.J. Roos), maier@biomed.au.dk (T.J. Maier).

44 J. Roos et al. / Pharmacology & Therapeutics 157 (2016) 43–64

Contents

Table 1Selected Wnt sign

Agent

OMP-18R5

OMP-54F28

LGK974IWP-L6SalinomycinNiclosamideNSC6680363289-8625FJ9XAV939JW55IWR-1PyrviniumPKF115-584, CGPNU-74654BC21iCRT3, iCRT5

iCRT14StAx peptidesResveratrolICG-001PRI-724

SAH-BCL9CWP232291

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442. The canonical Wnt pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453. The role of Wnt signaling in tumor cell growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454. Role of Wnt signaling in inflammatory disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455. Components of the Wnt signaling pathway as potential biomarkers of disease . . . . . . . . . . . . . . 456. Inhibitors of Wnt signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457. Non-steroidal anti-inflammatory drugs as Wnt pathway inhibitors . . . . . . . . . . . . . . . . . . . . 468. Introduction to the pro-inflammatory enzyme 5-lipoxygenase and its inhibitors . . . . . . . . . . . . . . . 479. Modulation by 5-LO and 5-LO inhibitors of Wnt signaling in leukemic stem cells . . . . . . . . . . . . . 4710. Conclusion and future direction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

44454547484951535557585858

1. Introduction

CanonicalWnt signaling is of pivotal importance in the pathogenesisof a number of cancer types and crucially affects tumor initiation, cancercell proliferation, cancer cell apoptosis, self-renewal of leukemic cellsand metastasis. We and others have shown that the Wnt/β-cateninsignaling pathway is the key signaling pathway inmany cancers, partic-ularly in those involving maintenance of cancer stem cells (CSC)(Muller-Tidow et al., 2004; Wang et al., 2010). CSCs represent a smallportion of the total tumormass in solid aswell as hematological cancers(Tirino et al., 2013). They are thought to persist as a distinct populationin the remaining tumor tissues after destruction of the bulk of tumormass by conventional chemotherapy. Theymight trigger disease relapseand metastasis by generating novel tumor cells (Tirino et al., 2013). Forthis reason, therapeutics specifically targeting cancer stem cells havethe potential for significant improvement of future cancer therapies.

Considerable efforts have been undertaken to identify novel thera-peutics that could interfere with oncogenic Wnt signaling (B. Chenet al., 2009). Decades have been spent by both the pharmaceutical in-dustry and academic researchers who have used high-throughput

aling inhibitors, their targets and current stage of development.

Target Current Stage

Fzd receptors Clinical trials (NCT01345201, NCNCT01957007, NCT01973309)

Wnt proteins Clinical trials (NCT01608867, NCNCT02092363, NCT02050178)

Porcupine Clinical trial (NCT01351103)Porcupine In vivo (zebrafish)LRP6 In vivo (xenograft model)LRP6 In vivo (xenograft model)Dvl In vivo (Xenopus embryos)Dvl In vivo (Xenopus embryos)Dvl In vivo (xenograft model)Tankyrase 1/2 In vitro (cancer cell lines)Tankyrase 1/2 In vivo (mice)Tankyrase 1/2, axin In vivo (zebrafish)CK1α In vivo (xenograft models)

P049090 β-Catenin/TCF In vivo (Xenopus embryos)β-Catenin/TCF In vivo (zebrafish)β-Catenin/TCF In vitro (HCT116 cell line)β-Catenin/TCF In vitro (colon cancer cell lines, p

colon cancer specimens)β-Catenin/TCF In vivo (xenograft models)β-Catenin/TCF In vitro (colorectal cancer cell linβ-Catenin/TCF antioxidant Clinical trial (NCT00256334, NCTCBP In vivo (xenograft model)CBP Clinical trials (NCT01606579, NC

NCT02195440, NCT01302405)β-Catenin/Bcl9 In vivo (xenograft model)Sam68 Clinical trial (NCT01398462)

screens to identify small-molecule inhibitors targeted to different Wntsignaling pathway constituents. Regrettably, only one compound,namely PRI-724 (Prism Pharma Co, Ltd/Eisai, Table 1 and Supplementa-ry Table 1), which directly disrupts the transcriptional co-factor func-tion of β-catenin has been developed and entered phase I clinical trials(Safety and Efficacy Study of PRI-724 in Subjects with Advanced SolidTumors; ClinicalTrials.gov Identifier: NCT01302405). Other Wnt-suppressive drug candidates interfere with Wnt signaling at the levelof the Wnt ligand binding or ligand-induced receptor activation. Someof these compounds were/are also included in recent clinical trials(see Section 6). However, the potential side-effects and selectivity ofthe suppressive effects of this class of agents on the canonicalWnt path-way still remain unclear.

Non-steroidal anti-inflammatory drugs (NSAIDs), such as sulindac,indomethacin, and celecoxib, are able to inhibit Wnt signaling at highconcentrations and several mechanisms have been proposed to explainthese effects (see Section 7). We recently found that these drugs, atsomewhat higher concentrations than those required to inhibit cycloox-ygenase (COX) enzymes, also suppress 5-LOproduct formation suggest-ing a novel mechanistic linkage between the 5-LO pathway and

References

T02005315, (Gurney et al., 2012)

T02069145, (Smith et al., 2013; Yeung et al., 2014)

(Liu et al., 2013)(X. Wang et al., 2013)(Mao et al., 2014; Wang et al., 2012)(W. Lu et al., 2011; Osada et al., 2011; Sack et al., 2011)(Shan et al., 2005)(Grandy et al., 2009)(Fujii et al., 2007)(Huang et al., 2009)(Waaler et al., 2012)(B. Chen et al., 2009)(Thorne et al., 2010; Wiegering et al., 2014)(Lepourcelet et al., 2004)(Trosset et al., 2006; Buikema et al., 2013; Pradhan & Olsson, 2014)(Tian et al., 2012)

rimary human (Gonsalves et al., 2011)

(Gonsalves et al., 2011)es) (Grossmann et al., 2012)00433576) (Chen et al., 2012)

(Emami et al., 2004; Arensman et al., 2014)T01764477, (El-Khoueiry et al., 2013)

(Takada et al., 2012)JW Pharmaceuticals

45J. Roos et al. / Pharmacology & Therapeutics 157 (2016) 43–64

oncogenic Wnt signaling (Roos et al., 2014). Furthermore, we andothers showed that pharmacological or genetic targeting of 5-LO effi-ciently interferes with Wnt signaling and maintenance of leukemicstem cells (LSCs) in chronicmyeloid leukemia (CML) and acutemyeloidleukemia (AML) (Roos et al., 2014).

In the present review, we aim to simplify this recent but rather com-plex subject in order to appeal to a larger number of readerships andmaximize the interest in the topic. We provide a brief introduction toWnt signaling and its role in tumorigenesis, as well as an overview ofavailableWnt inhibitors and theirmolecularmechanisms ofWnt signal-ing suppressionwith focus on drugs interferingwith eicosanoid biosyn-thesis. Emphasis is placed on the recently discovered role of 5-LO inregulating the canonical Wnt pathway. Finally, we discuss the possibleclinical implications of these findings for new stem-cell specific thera-peutics in oncology.

2. The canonical Wnt pathway

The Wnt signaling pathway is a highly conserved pathway with aprominent role in embryogenic development, adult tissue homeostasis,cell polarization, stem cell biology, cell differentiation, and proliferation(Nusse & Varmus, 1992; Cadigan & Nusse, 1997; van deWetering et al.,2002; Clevers, 2006; Clevers & Nusse, 2012). Traditionally, theWnt sig-naling pathway is separated into two routes; the non-canonical and thecanonical pathways (see Section 2.1). They differ in their dependencyon the protein β-catenin. The non-canonical pathways, such as the pla-nar cell polarity (PCP) and Ca2+-pathway, regulate processes such ascell movement, migration (Kuhl et al., 2001; Mikels & Nusse, 2006;Angers & Moon, 2009), cell orientation (Seifert & Mlodzik, 2007;Angers &Moon, 2009), and function through an β-catenin independentmechanism.

2.1. Overview of the canonical Wnt/β-catenin pathway

In the canonical pathway (Fig. 1),β-catenin is the key effector,whichis responsible for the transduction of the signal to the nucleus. At thissite, β-catenin triggers the transcription ofWnt-specific genes responsi-ble for the control of cell fate decisions in many cells and tissues(Valenta et al., 2012). In addition to its role in the Wnt signalingpathway, β-catenin is associated with cadherins such as E-cadherin. β-Catenin connects cadherins with the actin cytoskeleton (through α-catenin), forming adherents junctions. It is important for cell stability,cell migration and adhesion (Harris & Peifer, 2005; Wilusz & Majka,2008).

2.2. Regulation of the Wnt/β-catenin pathway

If not in complex with E-cadherin, β-catenin is targeted for rapiddegradation by the proteasome, a protein complex involved in the elim-ination of mainly dysfunctional cellular proteins (Fig. 1A) (Aberle et al.,1997). This breakdown is mediated by a multiprotein destruction com-plex which includes the tumor suppressor proteins, adenomatouspolyposis coli (APC) and axin, glycogen synthase kinase 3 (GSK3β)and casein kinase 1α (CK1α) (McDonald & Silver, 2009; Clevers &Nusse, 2012), together with the serine/threonine phosphatase A2

(PPA2) (Luo et al., 2007; Su et al., 2008). APC and axin function as a scaf-fold, facilitating GSK3β- and CK1α-mediated phosphorylation of criticalresidues within β-catenin (Kimelman & Xu, 2006; Roberts et al., 2011).In particular, CK1α phosphorylates β-catenin at Ser45, priming the se-quential phosphorylation of Thr41, Ser37, and Ser33 by GSK3β (Liuet al., 2002; Xing et al., 2003; Valenta et al., 2012; Stamos & Weis,2013). Subsequently, β-catenin interacts with the ubiquitin machineryvia its phosphorylated Ser33 and Ser37 residues. As a consequence, β-catenin is ubiquitinated and subsequently degraded by the 26S protea-some (Aberle et al., 1997; Hart et al., 1999). The phosphatase PP2A, alsoassociated with axin and/or APC, counteracts the phosphorylation-

induced degradation of β-catenin by directly dephosphorylating β-catenin at the critical residues important for proteasomal degradation.Furthermore, PP2A binds APC and is suggested to dephosphorylatethe APC protein leading to impaired affinity of APC to β-catenin(Seshacharyulu et al., 2013). Recently, Huang et al. discovered that thetwo poly-ADP-ribosylating enzymes, tankyrase 1 and tankyrase 2,bind to a highly conserved domain of axin and trigger its degradationthrough the ubiquitin–proteasome pathway, thereby, releasing β-catenin (Huang et al., 2009).

The Wnt/β-catenin pathway is activated when a secreted Wnt gly-coprotein binds to the N-terminal extracellular cysteine-rich domainof a Frizzled (Fzd) receptor (Fig. 1B). Overall, there are currently 19Wnt ligands and 10 Fzd receptors that have been identified in humans(Silva-Garcia et al., 2014). Upon binding of the Wnt ligand to Fzd, theco-receptor, low density lipoprotein receptor-related protein 5/6(LRP5/6), interactswith Fzd and initiates a cascade of events that resultsin disruption of the β-catenin destruction complex. As consequence, β-catenin is stabilized and is facilitated in its nuclear translocation. Activa-tion of the Fzd receptor leads to phosphorylation of disheveled (Dvl)which causes direct binding of Dvl to the receptor complex (Cruciat,2014). Dvl subsequently multimerizes and induces the formation ofthe so-called LRP-associated Wnt signalosome (Bilic et al., 2007). Thisprocess triggers LRP6 phosphorylation by CK1 (Davidson et al., 2005;Bilic et al., 2007). Phosphorylated LRP6 serves as a docking site foraxin (Mao et al., 2001; Tamai et al., 2004; Davidson et al., 2005; Zenget al., 2005) which recruits the β-catenin destruction complex tosignalosomes (Niehrs, 2012). Furthermore, phosphorylation of LRP5/6leads to suppression of GSK3β activity by direct binding of the enzymeto the co-receptor (Piao et al., 2008). Both events protect β-cateninfrom GSK3β-mediated phosphorylation, which allows β-catenin to ac-cumulate in the cytoplasm and translocate into the nucleus (Taelmanet al., 2010).

Nuclear β-catenin forms a complex with the members of the T-cellfactor/lymphoid enhancer factor (TCF/LEF) transcription factors. Thiscomplexed β-catenin acts as a transcriptional co-activator by displacingco-repressors like Groucho and histone deacetylases (HDACs) (Brantjeset al., 2001; Daniels & Weis, 2005; Archbold et al., 2012; Cadigan,2012) from TCF/LEF. Furthermore, complexed β-catenin recruits otherco-activators such as Bcl9/Legless (Lgs), Pygopus (Pyg) (Kramps et al.,2002; Parker et al., 2002; Thompson et al., 2002), and CREB-binding pro-tein (CBP)/p300 (Hecht et al., 2000; Takemaru&Moon, 2000) to the TCF/LEF transcription factor. These events trigger DNA binding and transcrip-tion of the target genes, including c-myc, cyclin D1 and axin 2 (Lustiget al., 2002; Davidson et al., 2005; Bilic et al., 2007). For further informa-tion, the following website offers an extensive list of Wnt target genes:http://www.stanford.edu/group/nusselab/cgi-bin/wnt/target_genes.

3. The role of Wnt signaling in tumor cell growth

Wnt signaling plays a pivotal role in tumor cell growth by regulatinga number of genes involved in programmed cell death (Pecina-Slaus,2010), cell cycle control, and angiogenesis (Clifford et al., 2008).

Gain or loss-of-function mutations of several members of the Wnt/β-catenin signaling pathway that lead to constitutive activation of β-catenin have been shown to have oncogenic effects in solid tumors(Peifer & Polakis, 2000; Giles et al., 2003). Mutations in β-cateninwere first uncovered in colorectal cancer (Rubinfeld et al., 1993; Suet al., 1993) and later – with a lower frequency – also in other humantumor entities (Peifer & Polakis, 2000; Giles et al., 2003; Luu et al.,2004). Furthermore, Wnt signaling plays a crucial role in the processof leukemogenesis (see Section 3.4).

3.1. Cell survival

Programmed cell death constitutes a fundamental cellular event in-volved in the regulation of various physiological processes (Lockshin &

Fig. 1. A simplified overview of theWnt/β-catenin signaling pathway. In the absence of a Wnt ligand (A), β-catenin is either bound to E-cadherins connecting them to the actin cytoskel-eton or bound in a multiprotein destruction complex, consisting of axin, APC, GSK3β, CK1α, PP1, and PP2A. In this complex, β-catenin is sequentially phosphorylated (P) at different Thrand Ser residues by CK1α and GSK3β. This phosphorylation creates a binding site for E3 ubiquitin ligase β-TrCP, leading to β-catenin ubiquitination and therefore, proteasomal degrada-tion. The transcription of theWnt target genes is repressed by binding of co-repressors (i.e. Groucho and HDACs) to the TCF/LEF transcription factor. The binding of aWnt ligand (B) to itsreceptors, Fzd and LRP5/6, results in the activation of Dvl. Dvl is then recruited to the receptor complexwhich leads to LRP6 phosphorylation (P) and recruitment of theβ-catenin destruc-tion complex to the receptors (currently, there is only evidence for binding of axin, APC, GSK3β, and CK1α to the receptor complex). Thereby, the phosphorylation of β-catenin is inhibited,allowing the free protein to accumulate in the cytosol and translocate to the nucleus where it can act as a co-activator for TCF/LEF-mediated transcription. For this, β-catenin replaces theco-repressors while recruiting various cofactors, including Bcl9/Lgs and Pygopus. APC: adenomatous polyposis coli proteins and; the kinases, GSK3β: glycogen synthase kinase 3β; CBP,CREB-binding protein; CK1α: casein kinase α; Dvl, disheveled; GRG, Groucho; HDAC, histone deacetylase; Fzd, Frizzled; LRP5/6, low density lipoprotein receptor-related protein 5/6;PP2A, phosphatase A2; Pyg, Pygopus; TCF/LEF, T-cell factor/lymphoid enhancer factor.

46 J. Roos et al. / Pharmacology & Therapeutics 157 (2016) 43–64

Zakeri, 2007). Among the various forms of programmed cell death, ap-optosis represents one of the best characterized and is evolutionaryhighly conserved (Galluzzi et al., 2015). Since tissue homeostasis is theresult of a tight balance between cell proliferation and cell death, evensmall alterations in the rate of apoptosis may significantly impact theoverall cell number (Fulda, 2009).

Apoptosis can be viewed as a safeguard mechanism to limit abnor-mal tissue growth and tumor formation. Consequently, cellular abilityto evade apoptosis is one of the hallmarks of cancers (Hanahan &Weinberg, 2011). A number of components of the Wnt signaling path-way such as Dvl, β-catenin, and GSK3β have been shown to modulateapoptosis (Li et al., 2006). Furthermore, β-catenin silencing experi-ments using siRNA approaches identified several Wnt target genes re-lated to apoptosis, including MYBL2, BAG2, BAG3, PTEN, HIF1A, andDAP3 as well as the anti-apoptotic protein Bcl-XL (Huang et al., 2006).Thus, in the majority of cancer cell types, Wnt signaling is consideredto promote cell survival and to inhibit apoptosis, though pro-apoptoticeffects due to activated Wnt signaling have also been reported(Benchabane & Ahmed, 2009). In this context, it should be noted that,in cancer, not only is apoptosis dysfunctional, but there is also an in-crease in the activity of survival pathways that promote proliferationand cell growth.

3.2. Cell cycle regulation

Besides resistance to apoptosis, many cancer cells exhibit dysfunc-tion of the cell cyclemachinerywith regard to the complex series of pro-tein–protein interactions, protein degradation, and phosphorylationevents that regulate the transition to the respective four cell cyclephases (i.e., G1, S, G2, M). The major players at these checkpoints arecyclin-dependent kinases (CDK) which are activated by forming com-plexes with transiently expressed cyclins.

As described above, a key step in the activation of theWnt pathwayis the formation of a complex between nuclear β-catenin and membersof the TCF/LEF family of transcription factors (van de Wetering et al.,1997). The continuous presence of the nuclear TCF/β-catenin complexfosters cancer formation by increasing expression of a number ofproto-oncogenes regulating cell growth, inhibition of apoptosis, andgenes affecting cell shape and cellmigration. Consistentwith these find-ings, disruption of TCF/β-catenin complex formation in colon cancercells hindered target gene activation and inhibited tumor cell growthin vitro (Tetsu & McCormick, 1999; van de Wetering et al., 2002). In-creased stability of β-catenin leads to expression of a number ofdown-stream target genes, such as the G1 cell cycle control regulators,cellular myelocytomatosis (c-myc), and cyclin D1 (Reya & Clevers,

47J. Roos et al. / Pharmacology & Therapeutics 157 (2016) 43–64

2005). C-myc is a proto-oncogene overexpressed in a variety of humancancers including leukemia (Dang, 1999) and plays a role in cell cycleprogression, apoptosis, and cellular transformation (Hoffman et al.,2002). Cyclin D1 is an oncogenic cyclin, which is frequentlyoverexpressed or amplified in breast, prostate, and colon cancers(Ortega et al., 2002). Nevertheless, some components of theWnt sig-naling cascade also function directly to promote formation of thespindle apparatus during mitosis (Niehrs & Acebron, 2012).

3.3. Tumor vascularization

A crucial role forWnt signaling in tumor vascularization is supportedby the presence of Wnt target genes that encode for pro-angiogenicfactors. In particular, the gene encoding for vascular endothelial growthfactor A (VEGF-A), a potent andwell-characterized pro-angiogenic pro-tein, is directly regulated by the TCF/β-catenin complex (Clifford et al.,2008) and is clearly associated with boosted tumor angiogenesis and apoor prognosis (Gratzinger et al., 2007).

3.4. Leukemic stem cell self-renewal

Although the involvement of β-catenin in carcinogenesis was firstdiscovered in solid tumors,β-catenin overexpression is also highly asso-ciated with several leukemic subtypes (Chung et al., 2002). Further-more, the dysregulation of the Wnt//β-catenin signaling pathway wassuggested to play an important role in the self-renewal of LSC in atleast AML and CML patients (Lane et al., 2011; Mochmann et al., 2011;Siapati et al., 2011; Scheller et al., 2013).

LSCs are typically rare and possess properties that are distinct frommost other tumor cells (Lapidot et al., 1994). These cells can undergoself-renewal to maintain an undifferentiated state. They are alsomultipotent, highly proliferative (Jordan, 2007), and may originatefrom normal hematopoietic stem cells (HSC) (Krivtsov et al., 2006;Eppert et al., 2011). Although the role of Wnt/β-catenin signaling inthe self-renewal of normal HSCs is under debate (Cobas et al., 2004;Jeannet et al., 2008; Koch et al., 2008), several groups have shown thatthe Wnt/β-catenin signaling pathway is required for the self-renewalof LSCs derived from either HSC or more differentiated granulocytemacrophage progenitors (GMP) (Muller-Tidow et al., 2004; Malhotra& Kincade, 2009; Wang et al., 2010; Luis et al., 2012). Progression,disease relapse, and drug resistance of leukemic cells depend on LSCsthat resist treatment. Aberrant activation of the Wnt/β-catenin self-renewal pathway has been identified to drive human blast crisis LSCpropagation (Jamieson et al., 2004; Abrahamsson et al., 2009). Sup-pression of β-catenin reverts the LSC stage to a pre-LSC-like stageand significantly reduces the growth of human leukemic cells(Yeung et al., 2010). The chemotherapeutic agents that are usedtoday effectively eradicate the blast cells, which represent partially dif-ferentiated, usually unipotent precursor cells. However, these agentshave very little effect on LSCs. Since the canonical pathway is inactivein most normal cells, inhibition of the Wnt/β-catenin pathway holdspromise for developing novel, targeted therapies for LSCs.

Finally, Wnt/β-catenin signaling pathways have been shown to beable to control proliferation, survival, and differentiation of hematopoi-etic cells (Reya et al., 2003). Taken together, the functional versatility ofWnt/β-catenin signaling is remarkable through its influences, on manydevelopmental levels, including organ and cell specification and differ-entiation as well as maintenance of stem cell activity.

3.5. Tumor prognosis and outcome

In the last couple of years evidence has been accumulating suggest-ing that an aberrant regulated Wnt signaling pathway is correlatedwith the clinical outcome of various cancers such as colorectal cancer(CRC) (Ting et al., 2013; Bruun et al., 2014; Aguilera et al., 2015;Nazemalhosseini Mojarad et al., 2015), AML (Fu et al., 2014; Kuhnl

et al., 2015), gliomas (Rossi et al., 2011; Wu et al., 2013; Guo et al.,2015), nasopharyngeal cancer (NPC) (Xu et al., 2013; Ren et al., 2015)and breast cancer (Dey et al., 2013). In particular, it could be shownthat overexpression of canonical Wnt signaling components is highlyassociatedwith the risk of developingmetastases in patientswith an ag-gressive formof breast cancer (Dey et al., 2013). Similar resultswere ob-tained in a study of potential prognostic factors in glioblastomapatients.

In this case, the investigators demonstrated thatβ-catenin is a highlypredictive marker of malignant behavior and short survival (Rossi et al.,2011). Aberrant expression of β-catenin and other components and tar-get genes of theWnt pathway, such as sFRP1,Wnt-2, E-cadherin, c-mycand LGR5, have also been correlated with poor prognosis and tumor ag-gressiveness inNPC, brainstem glioma, AML and thyroid cancer patients(Wu et al., 2013; Xu et al., 2013; Fu et al., 2014; Michelotti et al., 2015;Ren et al., 2015). Yet not only the expression level, but also the mem-brane/cytosolic or nuclear localization of β-catenin – which is a sign ofactivated Wnt signaling – can be a negative prognostic factor, as wasshown in patients with CRC (Bruun et al., 2014; NazemalhosseiniMojarad et al., 2015). Additionally, nuclear DKK1, localized at specificchromatin sites of active transcription, is associated with decreasedoverall survival rate and progression-free survival after chemotherapyadministration (Aguilera et al., 2015). Furthermore, single nucleotidepolymorphism (SNPs) in APC and β-catenin (CTNNB1) genes, whichare known to alter consensus splicing sites sequences, transposableelements and transcription factor binding sites, are correlated withoverall survival rates (Ting et al., 2013). Based on the pathophysiologicalimportance of theWnt signaling pathway in relation to tumor prolif-eration, suppression of apoptosis, migration, resistance to chemothera-py and maintenance of cancer stem cells (Roarty & Rosen, 2010;Vermeulen et al., 2010; Alison et al., 2012; Debeb et al., 2012), it is notsurprising that an aberrantly activated Wnt signaling pathway is, inmany cases, associated with poor prognosis of cancer patients. Takentogether, all these findings support the concept ofWnt signaling inhibi-tion as a feasible therapeutic strategy.

4. Role of Wnt signaling in inflammatory disorders

4.1. Disease association

Reports over the last decade have provided strong evidence fora pathophysiological role of Wnt signaling in a number of non-malignant classical inflammatory diseases. In particular, several studieshave substantiated a crucial role of Wnt-mediated signaling in the acti-vation of fibroblast-like synoviocytes during the pathogenesis of rheu-matoid arthritis, triggering destruction of the articular cartilage andbone (Sen, 2005). Furthermore, Wnt signaling pathway and Wnt regu-latory proteins, like Dickkopf, may trigger bony fusion in ankylosingspondylitis and thus, may serve as potential biomarkers of this disease(Corr, 2014).

Changes in the expression of several genes of the Wnt signalingpathway were found to be associated with impaired lung function inchildrenwith asthma andmay contribute to the pathogenesis of the dis-ease (Sharma et al., 2010). Further mechanistic insight was recentlyprovided by Trischer et al. who showed that Wnt signaling via theWnt10b ligand is an important modulator of T cell activation duringasthma pathogenesis (Trischler et al., 2015). Guo et al. demonstratedthat intratracheal administration of the Wnt ligand, Wnt3a, or an anti-body raised against Dickkopf-1 activating Wnt signaling in alveolar ep-ithelial cells suppressed macrophage and neutrophil infiltration intolungs (Guo et al., 2015). Pathophysiological roles of Wnt signalinghave also been identified in chronic obstructive pulmonary disease(COPD) where increased β-catenin activity in fibroblasts was found tobe relevant for the diseases-associated remodeling of the extracellularmatrix (Baarsma et al., 2011). Interestingly, over-activity of Wnt signal-ing in primary bronchial epithelial cells sensitized individuals to

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cigarette smoke-induced inflammation, potentially triggering COPD(Heijink et al., 2013).

A crucial role of Wnt signaling pathway components is also evidentin inflammatory bowel disease (IBD) and the transition to themalignantstage. Several studies found a stage-specific increased or decreasedexpression of a number of Wnt pathway-related genes, including Wntligands, Fzd and Dickkopf-4, in colonic biopsies of subjects with ulcera-tive colitis or in IBD-associated colorectal neoplasia as well as in cancers(You et al., 2007, 2008; Claessen et al., 2010).

The literature also supports a role of Wnt signaling in a number ofother diseases featuring an inflammatory component. Malgor et al. re-cently provided evidence for an increase in the protein levels of theWnt5a ligand in advanced arterial arteriosclerotic lesions, whencompared with less advanced arterial lesions. Serum levels of Wnt5awere also significantly higher in patients suffering from atherosclerosiscompared to healthy controls, suggesting that Wnt signaling enhancesthe process of atherosclerotic plaque formation at a late stage (Malgoret al., 2014). Polymorphism or reduced expression of the secretedFrizzled-related protein (sFRP)3 has also been observed in patientswith osteoarthritis (Loughlin et al., 2004) and Wnt4 ligand inductionhas been related to kidney damage or polycystic kidney disease(Rodova et al., 2002; Surendran & Simon, 2003; Terada et al., 2003).

Importantly, Wnt signaling appears to play a pathophysiologicallyrelevant role in neurodegenerative diseases, asWnt-mediated neuroin-flammation triggers infiltration of leukocytes and production of pro-inflammatory mediators in the central and peripheral nervous systems,contributing to neuronal cell damage or death as well as to generationand maintenance of chronic pain (Yuan et al., 2012; Ji et al., 2014).

Wnt is an important regulator of neuroinflammation in the braincontrolling the dialog between neurons and glia or glial and neuralstem/progenitor cells (NPCs) during the inflammatory processes(Marchetti & Pluchino, 2013). The expression of Wnt ligands and Wntsignaling components has only been identified in astrocyte and macro-phage/microglia (Halleskog et al., 2011; L'Episcopo et al., 2011) that canharbor Wnt receptors and respond to Wnt in either pro- or anti-inflammatorymanner (L'Episcopo et al., 2014a, 2014b). Indeed, deregu-lation of Wnt/β-catenin signaling pathway, dysregulation of Wnt/Fzdcascade and increase in inflammatory processes have been proposedas common determinants in some neurodegenerative events of schizo-phrenia (Miyaoka et al., 1999), Parkinson's (PD)/Alzheimer's Disease(AD) (De Ferrari & Inestrosa, 2000; Chong et al., 2007; De Ferrari et al.,2007; Varela-Nallar et al., 2009; Inestrosa & Arenas, 2010; Kim et al.,2011; Purro et al., 2012) and autistic syndrome (Cao et al., 2012).

Wnt1/β-catenin signaling is relevant in maintaining the integrity ofdopaminergic neurons by blocking GSK3β-induced phosphorylationand proteasome degradation of β-catenin. Stabilized β-catenin cantranslocate into the nucleus and associate with transcription factorsto regulate the expression of Wnt target genes (Rawal et al., 2009;Inestrosa & Arenas, 2010).

Furthermore, β-catenin levels are markedly reduced in AD pa-tients carrying presenilin-1-inherited mutations (Zhang et al.,1998) and it has been suggested that similar mutations may disturbβ-catenin translocation to the nucleus (Nishimura et al., 1999), likelyaffecting Wnt activity. In this context it is relevant that Aβ has beenreported to inhibit Wnt signaling by directly binding to the Frizzledreceptors (Magdesian et al., 2008) and to LRP-5 or LRP-6 leading tothe inhibition of GSK3β through a cascade of intracellular reactionsthat involve protein kinases and adaptor proteins (Toledo &Inestrosa, 2010). In addition, genetic studies revealed that LRP6polymorphisms are causally linked to AD (De Ferrari & Inestrosa,2000; De Ferrari et al., 2007). Collectively, the literature of the pastdecade suggests a pathophysiologically relevant modulatory role ofcomponents of the Wnt signaling pathway in a number of classicalinflammatory or inflammation-associated diseases including rheu-matoid arthritis, bronchial asthma, COPD; IBD, atherosclerosis, anky-losing spondylitis and neuroinflammatory diseases.

4.2. Connection with inflammatory signaling pathways

Themechanistic link betweenWnt signaling and classical inflamma-tory pathways, however, is only poorly understood. Yu et al. showedthat Wnt5a and Wnt3a ligands are capable of directly triggering pro-inflammatory cytokine production from murine macrophages in someway through the Toll-like receptor (TLR) 4 (C.H. Yu et al., 2014).Conversely, activation of TLR signaling in alveolar epithelial cells byMycobacterium bovis, Bacillus Calmette–Guerin or purified lipopolysac-charide led to suppression of Wnt/β-catenin signaling and decreasedtranscriptional activity of nuclear β-catenin complexes. Notably, Wnt4ligands were recently shown to prevent aging-related bone loss andosteoporosis in an animal model by inhibiting pro-inflammatorynuclear factor-κB (NF-κB) signaling (B. Yu et al., 2014). Very recentmechanistic studies support a role of the Wnt pathway in promotingpro-inflammatory IFN-γ signaling and T cell differentiation in the trig-gering of ulcerative colitis (Sato et al., 2015). Thus, taken together,these recent data suggest an important role of oncogenicWnt signalingin regulating inflammatory processes.

This mechanistic linkage sheds new light on the previously well-recognized, but poorly understood role of chronic inflammation as amajor risk factor in tumorigenesis. The recent findings indicate thatchronic inflammation may facilitate carcinogenesis by enhancing infil-tration of immune cells and promoting stromal remodeling and thiscould be maintained by activation of the Wnt signaling pathway(Hanahan&Coussens, 2012; Briso et al., 2013). Conversely, inflammato-ry processesmay trigger activation ofWnt signaling, thereby enhancingtumor cell proliferation. This may explain, at least partially, whycolorectal cancer tends to develop on the background of chronic inflam-matory bowel disease (Guina et al., 2015). Cutaneous squamous cellcarcinomas are also considered to develop on the basis of underlyinginflammatory skin diseases, such as lupus vulgaris (Baldursson et al.,1993; Motswaledi & Doman, 2007). Thus, therapeutics that efficientlytarget Wnt signaling may act to curtail the onset of a series ofinflammation-dependent disorders, including classical inflammatorydiseases, neurodegenerative diseases as well as malignancies.

5. Components of the Wnt signalingpathway as potential biomarkers of disease

An increasing number of studies document the use of components ofthe Wnt signaling pathways as possible biomarkers of the stage, sever-ity and progression of severalmalignant diseases. LEF-1may function asa potential marker of malignant transformation to aggressive gliomas(Pecina-Slaus et al., 2014). TheWnt5A ligand was found to be a predic-tive marker of survival in human cervical cancer (Lin et al., 2014), andhigh serum levels of Dickkopf-1 reliably indicated a poor prognosis inprostate cancer (Rachner et al., 2014). Furthermore, serum DKK1 levelswere significantly lower in patients with lung cancer but rapidly nor-malized after chemotherapy (Xu et al., 2014). The G-protein coupled re-ceptor LGR5 regulating Wnt signaling may also function as a potentialprognostic biomarker of colorectal cancer (He et al., 2014) and Fzd re-ceptors may represent novel surface markers for an aggressive type ofneuroblastoma cells with stem-cell like features (Cantilena et al., 2011).

Wnt components, such asβ-catenin, not onlymayprovide an indica-tion of stage and severity of the disease, but also may have potential aspredictive therapeutic markers of chemoresistance and radioresistancein cutaneous squamous cell carcinoma (CSCC) (Y. Zhang et al., 2014).Notably, Wnt components may be useful for monitoring stage andprogression of some non-malignant diseases, such as serum levels ofDickkopf-1 to assess inflammatory processes during ankylosing spon-dylitis (Yucong et al., 2014) or abnormalities of bone metabolisms inchronic kidney disease (Thambiah et al., 2012) and in patients withfluorine bone injury (W. Wang et al., 2013). Furthermore, LRP5 hasproven to be a crucial player in murine lung fibrosis and is a marker ofdisease progression and severity in subjects with idiopathic pulmonary

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fibrosis (Lam et al., 2014). It is reasonable to speculate that Wnt path-way biomarkers might well be useful for monitoring the efficacy ofWnt-targeting therapeutics (Serafino et al., 2014), as is true for urinaryLTE4 as amarker of anti-LT therapeutics (Chiu et al., 2014) or the urinaryPGE2 metabolite, PGE-M, to monitor pharmacological COX inhibition(Murphey et al., 2004). Future studies are needed to investigate theeffects of the different anti-Wnt therapeutics (see following section)on the diverseWnt pathway components and to identify possible candi-date biomarkers.

6. Inhibitors of Wnt signaling

Considering the enormous impact of canonical Wnt signaling oncancer development and other Wnt-related diseases, like fibrosis,neurological diseases, osteoporosis and regenerative medicine (seeSections 3 and 4), it is not surprising that intensive efforts have been un-dertaken to understand the fundamental processes and to discovermodulators of this pathway (Barker & Clevers, 2006; Verkaar &Zaman, 2011; Niehrs, 2012; Pattabiraman & Weinberg, 2014). Overthe last 30 years, a number of druggable components and protein–protein interactions of the Wnt pathway have been identified (targetsof selected Wnt pathway inhibitors are illustrated in Fig. 2). However,efficient targeting of Wnt signaling still remains in its infancy (Verkaar& Zaman, 2011; Anastas & Moon, 2013; Kahn, 2014).

In this section, we briefly review the most promising known targetsand Wnt pathway inhibitors, the latter consisting of small molecules,peptides, and antibodies (see Fig. 2, Table 1, and SupplementaryTable 1). The reader is also referred to several comprehensive reviews

A

Fig. 2.Mode of action of different inhibitors of Wnt signaling. Agents targeting different pathwadenomatous polyposis coli proteins and; GSK3β: glycogen synthase kinase 3β; CBP, CREB bdeacetylase; Fzd, Frizzled; LRP5/6, low density lipoprotein receptor-related protein 5/6; PP2Atankyrase.

on this topic for further thorough reading (Barker & Clevers, 2006;Verkaar & Zaman, 2011; Kahn, 2014; Le et al., 2015).

6.1. Inhibitors acting at the plasma membrane

The Wnt pathway (see Figs. 1 and 2) can be directly targeted at thecellmembrane, using antibodies against Fzd receptors andWntproteins(Rhee et al., 2002; He et al., 2004, 2005; You et al., 2004; Mikami et al.,2005; DeAlmeida et al., 2007; Gurney et al., 2012).

The monoclonal antibody, OMP-18R5, binds to five distinct Fzd re-ceptors (Fig. 2) through a conserved epitope and prevents binding ofWnt ligands (Gurney et al., 2012; Le et al., 2015). The therapeutic effectof this antibody was assessed in human xenograft models displayinggrowth inhibition of different tumor types (Gurney et al., 2012). OMP-18R5 is currently in phase I clinical trials as a single agent or for combi-nation therapy (see Table 1).

A very promising candidate is the antibody-based inhibitor OMP-54F28, a truncated Fzd8 receptor fused to the immunoglobulin Fc region(Le et al., 2015). OMP-54F28 inhibits Wnt signaling and tumor growthof a mouse mammary tumor virus (MMTV)-Wnt-1-induced tumorsand patient-derived cancer xenograft models (Smith et al., 2013;Yeung et al., 2014). OMP-54F28 is currently in a phase I clinical trialand further trials in combination with chemotherapeutics have beeninitiated (see Table 1).

Wnt maturation and secretion are specifically dependent on the O-acyltransferase, porcupine, which palmitoylates immature Wnt pro-teins. Inhibition of this enzyme by the selective and orally bioavailableinhibitor, LGK974 (Table 1 and Supplementary Table 1), prevented se-cretion of Wnt ligands and displayed good efficacy in murine and rat

B

ay components are displayed in purple boxes. For detailed description see Section 6. APC:inding protein; CK1α: casein kinase α; Dvl, disheveled; GRG, Groucho; HDAC, histone, phosphatase A2; Pyg, Pygopus; TCF/LEF, T-cell factor/lymphoid enhancer factor; TNKS,

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cancer models at well-tolerated doses (Liu et al., 2013). LGK974 is cur-rently in phase I clinical trials for patients with Wnt-dependent malig-nancies. Inhibitors of Wnt production (IWP) represent a differentchemotype of potent porcupine inhibitors (B. Chen et al., 2009; Dodgeet al., 2012). IWP-L6 (Table 1 and Supplementary Table 1), which in-hibits porcupine at subnanomolar concentrations, prevented posterioraxis formation in zebrafish (X. Wang et al., 2013).

Several compounds have been described to act at the level of theWnt co-receptor, LRP6 (Gupta et al., 2009; M. Chen et al., 2009; D. Luet al., 2011;W. Lu et al., 2011; Lu et al., 2014). The potassium ionophoresalinomycin, a natural product from the fungus Streptomyces albus,blocks Wnt-induced phosphorylation of LRP6 and promotes its degra-dation. Along with reduced β-catenin levels, salinomycin decreasedthe expression ofWnt target genes. The activity of this agentwas furtherdemonstrated in different cancer cell lines and in in vivo studies (Maoet al., 2014; Wang et al., 2012). A similar mode of action was describedfor the FDA-approved antihelminthic drug, niclosamide. Niclosamideexhibited antiproliferative activity in several cancer cell lines at lowmi-cromolar concentrations and inhibited tumor growth and livermetasta-sis formation in mice (W. Lu et al., 2011; Osada et al., 2011; Sack et al.,2011). However, none of these LRP-targeting agents has entered clinicaltrials for cancer treatment so far.

A different approach to target the Wnt pathway at the cell mem-brane is to disrupt the Dvl-Fzd protein–protein interaction (PPI),which is essential for the transduction of the Wnt signal from Fzd toDvl. In-silico screenings revealed three compounds, NSC668036,3289–8625, and FJ9, that binddirectly to the PDZdomain ofDvl, therebydisabling the Dvl–Fzd interaction (Table 1 and Supplementary Table 1).These compounds inhibited Wnt-dependent signaling in Xenopus ormouse xenograft models (Shan et al., 2005; Fujii et al., 2007; Grandyet al., 2009).

6.2. Inhibitors acting at the level of axin

Another promising strategy to inhibitWnt signaling is the regulationof axin stability. The poly-ADP-ribosyltransferases (PARPs), tankyrases1/2, were shown to destabilize axin through PARsylation leading to sub-sequent proteasomal degradation.

Thus, tankyrase inhibitors stabilize axin, consequently enhance deg-radation of β-catenin, and suppress the growth of colon cancer cell lines(Huang et al., 2009; Lehtio et al., 2013).

Several chemotypes were described as tankyrase inhibitors, includ-ing XAV939, IWR-1, and JW55 (B. Chen et al., 2009; Huang et al.,2009; Waaler et al., 2012) (Table 1 and Supplementary Table 1). IWR-1 was active in zebrafish and JW55 reduced the growth of adenomasin mice (B. Chen et al., 2009; Waaler et al., 2012). Despite severalattempts to develop promising tankyrase inhibitors, none of them hasentered clinical trials so far.

The FDA-approved antihelminthic drug, pyrvinium pamoate(Table 1 and Supplementary Table 1), was described as a Wnt pathwayinhibitor in Xenopus laevis egg extracts (Thorne et al., 2010). Pyrviniumallosterically activates CK1α, leading to stabilization of axin and degra-dation of β-catenin, Pyg, and TCF/LEF. It showed a growth suppressiveeffect on various colon cancer cell lines and delayed tumor growthin vivo (Wiegering et al., 2014).

6.3. Inhibitors of β-catenin interactions

Interrupting the PPI of β-catenin in the nucleus has been a temptingbut demanding task (Hahne & Grossmann, 2013; Milroy et al., 2014).

A number of in vitro and in silico high-throughput screens revealeddifferent chemical scaffolds as disrupters of the TCF/β-catenin interac-tion, among them the natural fungal products, PKF115-584 andCGP049090 (Lepourcelet et al., 2004; Trosset et al., 2006; Z. Chenet al., 2009;Gonsalves et al., 2011; Tian et al., 2012) (Table 1 and Supple-mentary Table 1). Their activitywas demonstrated in colon and prostate

cancer cell lines, as well as in Xenopus embryos. These compounds werealso reported to disturb the β-catenin/APC interaction (Lepourceletet al., 2004).

An in-silico screen yielded PNU-74654 (Table 1 and SupplementaryTable 1) that selectively binds to β-catenin when applied in nanomolarconcentrations (Trosset et al., 2006). The biological activity of this com-pound has been described in zebrafish, during cardiomyocyte differen-tiation, and in adrenocortical tumors (Durand et al., 2011; Buikemaet al., 2013; Pradhan & Olsson, 2014).

Similarly, BC21 (Table 1 and Supplementary Table 1), a copper-containing agent, was identified in a virtual screen, and binds to β-catenin thus disrupting its interaction with TCF4. BC21 decreased theviability and colony formation of HCT-116 cells at low micromolarconcentrations (Tian et al., 2012). However, at higher concentrations,BC21 also targets protein phosphatase 2C and the proteasome (Danielet al., 2004; Rogers et al., 2006).

A high-throughput screen, employing an RNAi-based luciferase re-porter gene assay in Drosophila cells, identified the oxazole- andthiazole-based compounds, iCRT3, iCRT5, and iCRT14 (Table 1 and Sup-plementary Table 1)whichwere shown to disrupt theβ-catenin/TCF in-teraction in vitro and decrease Wnt target gene expression (Gonsalveset al., 2011). iCRT14 demonstrated modest activity in xenograft models(Gonsalves et al., 2011). Moreover, hydrocarbon-stapled peptides (StAxpeptides) have been reported to bind directly to β-catenin and specifi-cally disable its interaction with TCF4. These peptides selectivelyinhibited the Wnt pathway without influencing other developmentalpathways, such as Notch, Hedgehog, BMP, and transforming growthfactor-β (TGF-β) (Grossmann et al., 2012). Another stapled peptide,SAH-BCL9, selectively targets the β-catenin/Bcl9 interaction. SAH-BCL9blockedWnt-dependent gene transcription anddecreased proliferation,angiogenesis, and cellular migration in cell culture assays. Moreover,SAH-BCL9 demonstrated promising efficacy in colorectal carcinomaand multiple myeloma mouse xenograft models (Takada et al., 2012).

Some polyphenols, like quercetin and resveratrol (Table 1 and Sup-plementary Table 1), which are known for their polypharmacology,have also been reported to disrupt the β-catenin/TCF complex, to re-duceWnt target gene expression, and to show activity against differentcancer cells (Park et al., 2005; Chen et al., 2012). Resveratrol has alreadybeen studied in phase I clinical trials in patients with colon cancer (re-sults are pending).

A different approach to address canonical Wnt signaling at the levelof nuclear gene transcription is to prevent β-catenin interaction withthe transcriptional coactivator CBP in the nucleus. Emami et al. screeneda secondary structure-templated compound library for inhibition of theTOPFLASH reporter. The most promising hit, ICG-001 (Table 1 andSupplementary Table 1), selectively binds to CBP but not to the relatedp300 and competes with β-catenin for binding to CBP (Emami et al.,2004; Arensman et al., 2014). ICG-001 decreased Wnt target gene ex-pression and selectively inhibited the growth of colon cancer cell lines.In addition, a water-soluble form of ICG-001 reduced tumor volumesin mouse xenografts (Emami et al., 2004).The most promising hit, ICG-001 (Table 1 and Supplementary Table 1), selectively binds to CBP butnot to the related p300 and competes with β-catenin for binding toCBP (Emami et al., 2004; Arensman et al., 2014). ICG-001 decreasedWnt target gene expression and selectively inhibited the growth ofcolon cancer cell lines. In addition, a water-soluble form of ICG-001 re-duced tumor volumes in mouse xenografts (Emami et al., 2004).

Further structural development of β-catenin/CBP interaction inhibi-tors led to PRI-724, which is currently being assessed in phase I/II clini-cal trials for different types of cancers and liver cirrhosis (El-Khoueiryet al., 2013; Lenz & Kahn, 2014).

Interestingly, the activation of the nuclear receptors for retinoic acidand vitamin D by the respective ligands inhibits Wnt signaling. Thisinfluence was attributed to interaction of the nuclear receptors withβ-catenin, which sequester β-catenin away from TCF or coactivatorslike p300 and CBP (Takada et al., 2012).

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6.4. Additional targets and agents

Further small molecules and natural products have been describedto modulate Wnt signaling (Jaiswal et al., 2002; Kim et al., 2006; Z.Chen et al., 2009; Albring et al., 2013; Zeller et al., 2013). JWPharmaceu-ticals is developing a small molecule, CWP232291 (Table 1 and Supple-mentary Table 1), that targets Sam68 (Src-associated in mitosis), aprotein that forms a complex with APC and regulates alternative splic-ing of TCF1, thereby affecting Wnt signaling (Morishita et al., 2011;Sebio et al., 2014). This agent also modulates NF-κB signaling and alter-native splicing of the anti-apoptotic protein Bcl2 (Kahn, 2014).CWP232291 is currently undergoing a phase I clinical trial in patientswith acute myeloid leukemia (Pattabiraman & Weinberg, 2014).

In addition, NSAIDs such as celecoxib, aspirin, and sulindac exhibitchemopreventive properties owing to their reduction of the synthesisof prostaglandin E2 (PGE2) which has an effect on β-catenin stability(Dihlmann et al., 2001; Grosch et al., 2001; Maier et al., 2005). Potentialmolecular mechanisms of interference with Wnt signaling by NSAIDsare discussed in more detail in the following section.

Although druggable components of the Wnt pathway exist andpotent agents have been developed, only a few drug candidates havereached the clinic so far and no convincing results are available as yet.Apart from off-target effects and poor pharmacokinetic properties, thesluggish progress in the field of Wnt-targeted therapies can be attribut-ed to a variety of issues (Anastas &Moon, 2013; Kahn, 2014). Firstly, theWnt signaling network is very complex and crosstalk occurs with vari-ous pathways. Thus, Wnt signaling components may have redundantfunctions and/or may be regulated by other pathways. Secondly, theWnt pathway is essential for embryonic development as well as fortissue homeostasis and repair in adults. Therefore, it is not surprisingthat the systemic application of Wnt inhibitors is limited by on-targettoxicity which might affect hematopoiesis and normal bone and intes-tine functions. Thus, specificity of action and determination of theappropriate therapeutic window are of major importance to achieve atolerable balance between anti-tumorigenic effects and possible ad-verse side effects. Increased understanding of Wnt pathway-relatedmutations and their effects in patients or the introduction of predictivebiomarkers for patient selectionwill facilitate the choice of an agent thatacts upstream or downstream of the destruction complex and mayprove crucial to successful targeting of the Wnt pathway. Thirdly,aberrantWnt signaling does not necessarily correlate with poor patientsurvival in all types of cancer (Kahn, 2014). Therefore, Wnt-inhibitingdrugs must be carefully chosen based on the origin and genesis of thetargeted disease. Moreover, Wnt signaling has been linked to cancerchemoresistance and combination of Wnt pathway inhibitors andconventional chemotherapeutics is considered a promising approachto cancer treatment (Anastas & Moon, 2013; Kahn, 2014). Despitethese hurdles that still have to be addressed in the future, and theneed for improved insight into the clinical applicability of Wnt inhibi-tors, pharmacologicalmodulation ofWnt signaling remains a promisingstrategy for the treatment of various diseases, especially cancer. In thisregard, already approved drugs, like 5-LO and COX inhibitors, thatinfluence Wnt signaling and thus, provide an acceptable therapeuticwindow, are likely to be of great value for future research on Wnt-targeted therapies.

7. Non-steroidal anti-inflammatorydrugs as Wnt pathway inhibitors

As far back as the early 1980s, evidence was generated showing thatlong term treatment of cancer patients with NSAIDs causes regressionor stagnation of tumor progression (Waddell & Loughry, 1983;Waddell et al., 1983). Large retrospective and prospective studies haveconfirmed these findings for different NSAIDs, such as aspirin (Thunet al., 1991; Burn et al., 2011; Ng et al., 2015), celecoxib (Steinbachet al., 2000; Phillips et al., 2002), sulindac (Labayle et al., 1991;

Giardiello et al., 1993), and exisulind (Arber et al., 2006) in colon cancerpatients, mainly in familial adenomatous polyposis (FAP) patients. Fur-thermore, animal studies using ApcMin mice (a mouse model forhuman FAP) also indicate that these NSAIDs provide protective effectsagainst cancer (Boolbol et al., 1996; Chiu et al., 1997; Wechter et al.,1997; Mahmoud et al., 1998a, 1998b).

NSAIDs are classified as non-selective or selective inhibitors of COX-1 and/or -2 (Fig. 4B). COX-1, on the one hand, is constitutively expressedin a wide range of tissues and considered to maintain tissue homeosta-sis, platelet aggregation, and cell signaling in health. The immediateearly gene COX-2 is predominantly induced by a number of inflamma-tory cytokines in inflamed tissues and during cellular stress. COXs arekey enzymes in the biosynthesis of a series of (patho)physiologically ac-tive prostanoids, such as PGE2, PGD2, prostacyclin, and thromboxane A2.COX-2-derived PGE2 triggers induction of fever, inflammation-relatedvasodilatation, and hyperalgesia but also has well-documentedmitogenic effects on different cancer cells. However, COX-1-derivedPGE2 maintains mucosal integrity of the gastro-intestinal system byimpairing gastric acid secretion and increasing gastric mucus secretion(Simmons et al., 2004).

In numerous cancer types, the mechanisms controlling COX-2 ex-pression are abrogated, leading to overexpression of COX-2 proteinand enhanced PG production (Eberhart et al., 1994; Hida et al., 1998;Achiwa et al., 1999; Chan et al., 1999; Mohammed et al., 1999; Molinaet al., 1999; Soslow et al., 2000; Khan et al., 2001; Kulkarni et al.,2001). Especially in colon cancer, upregulation of COX-2 has beenshown to occur already at the early adenoma stage and is, therefore,one of the earliest mechanisms deregulated during tumor development(Shiff & Rigas, 1999; Dixon, 2003). Thus, enhanced COX-2 expressionhas been attributed a key role at early stages of cancer developmentby promoting cell division (Chinery et al., 1999; Fosslien, 2000),inhibiting apoptosis (Sheng et al., 1998; Lin et al., 2001; Nzeako et al.,2002; Tang et al., 2002), altering cell adhesion, and enhancingmetasta-sis (Tsujii et al., 1997; Tomozawa et al., 2000; Chen et al., 2001; Kakiuchiet al., 2002; Li et al., 2002) and neovascularization (Liu et al., 1999;Leahy et al., 2000).

As mentioned previously, several clinical studies have shown thatlong term administration of NSAIDs protects against colon cancer, espe-cially in FAP patients and ApcMin mice. FAP patients are characterizedbymutations in the APC gene, which is one of themain binding partnersfor β-catenin (see Section 2), and is required for the degradation of β-catenin by the ubiquitin–proteasome pathway (see Fig. 1). Therefore,clinical data suggest that the cancer-protective effect of NSAIDS isboth due to their COX-inhibiting potential as well as to interferencewith the Wnt/β-catenin pathway, independently of COX. Additionally,it has been reported that interdependency also exists between thePGE2- and Wnt-signaling pathways, which is not only a characteristicof colon cancer but is also conserved in mammalian hematopoieticstem cells and is important for tissue regeneration (Goessling et al.,2009).

7.1. Crosstalk between prostaglandinE2 and the Wnt/β-catenin signaling pathway

One possibility for NSAIDs to interfere with the Wnt/β-cateninpathway is by inhibiting COX-2 activity. As Fig. 3 illustrates, crosstalkexists between PGE2 and β-catenin via the binding of PGE2 to itsheterotrimeric guanine nucleotide-binding protein (G protein)-coupledreceptors, EP2 and EP4, at the surface of the cell (Fujino et al., 2002).Sonoshita et al. demonstrated that homozygous deletion of the geneencoding EP2 causes a decrease in the number and size of intestinalpolyps in Apc (Delta 716) min mice, whereas homozygous deletion ofEP1 or EP3 had no effect on polyp formation in these mice (Sonoshitaet al., 2001). Binding of PGE2 to EP2/4 provokes the release of activatedGαs subunit which binds to axin through its regulator of G-proteinsignaling (RGS)-domain. The binding of Gαs to axin causes the release

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of GSK3β from the β-catenin complex. Furthermore, Gαs activatesadenylate cyclase, leading to the formation of cyclic adenosinemonophosphate (cAMP) and activation of protein kinase A (PKA)(Fujino et al., 2002). PKA, in turn, phosphorylates β-catenin at Ser675,thereby preventing its degradation by the proteasome pathway (Hinoet al., 2005). Additionally, recruited Gβ/γ activates phosphatidylinositol3-kinase (PI3K), which subsequently stimulates and phosphorylatesprotein kinase B (PKB/Akt) through protein dependent kinase 1(PDK1). PKB/Akt causes the phosphorylation and thereby, inactivationof GSK3β, which leads to stabilization and nuclear translocation of β-catenin (Castellone et al., 2005). In the nucleus, β-catenin bindstogether with TCF/LEF to the promoter regions of several genes whichare involved in proliferation, invasion, angiogenesis, survival, or thedevelopment of CSC and other tumor cells (Fig. 3). Consequently,inhibition of COX-2 activity by NSAIDs thwarts these effects, whichmay account for the anti-carcinogenic influence of these substances(Kaur & Sanyal, 2010; Vaish & Sanyal, 2012; S. Zhang et al., 2014).

However, recent studies provided strong evidence suggesting thatin addition to COX-2 inhibition, COX-2-independent mechanismscontribute to the anti-tumorigenic effects of these drugs. Apparently,

Fig. 3.Modes of action of NSAIDs on the Wnt signaling pathway. In the absence of a Wnt stimcomplex” that includes the tumor suppressors, axin and adenomatous polyposis coli (APC), the Sβ-TrCP.WhenWnt binds to the surface receptors, Frizzled and LRP5/6 (low-density lipoproteincatenin degradation complex, consisting of adenomatous polyposis coli (APC), axin, and glycoB) which phosphorylates and thereby inactivates GSK3βb (Cohen & Frame, 2001). Other proteiPKG inhibits β-catenin expression on the transcriptional level and blocks its transcriptional trCrosstalk between EP2 receptors and the β-catenin pathway: Binding of PGE2 to EP2 receptconcomitantly binds axin through its regulator of G-protein signaling (RGS)-domain. The bininactivated by PKB, which leads to stabilization and nuclear translocation of β-catenin. Gαs aPKA. PKA stabilizes β-catenin by phosphorylation. In the nucleus, β-catenin binds, togetherinvasion, angiogenesis, survival or the development of cancer stemcells (CRC). Transcriptional alike NF-κB and PPARγ/RXRα leading to a repression of β-catenin/TCF-dependent genes. A numproteins at different levels of theWnt signal transduction cascade. APC, adenomatous polyposiscyclooxygenase; Dvl, disheveled; FOXO4, Forkhead-Box-Protein 4; Fz, Frizzled; GRG, Grouchlipoprotein receptor-related protein 5/6; mPGES1, microsomal prostaglandin E2 synthase 1; PDPK A/B/G, protein kinase A/B/G; PP2A, protein phosphatase 2A; PPARγ, Peroxisome proliferatretinoid X receptor.

each NSAID influences a more or less separate COX-independent target(Grosch et al., 2001; Grösch et al., 2003; Soh &Weinstein, 2003; Gröschet al., 2005, 2006). NSAIDs also influence the Wnt/β-catenin pathwaythrough a number of COX-independent targets. Some of these targetsand related molecular mechanisms leading to interference with theWnt/β-catenin pathway are described below and illustrated in Fig. 3.The mechanisms of suppression of canonical Wnt signaling by 5-LO in-hibitors are described in Section 9.

7.2. Interference of protein kinaseGwith the Wnt/β-catenin signaling pathway

In human colon cancer cells, sulindac and its metabolite, sulindac sul-fone (exisulind), block the activity of guanosine 3′,5′-monophosphate(cGMP)-specific phosphodiesterases (PDE) 2 and 5, thus, leading tosustained increases in cellular levels of cGMP and activation of cGMP-dependent protein kinase (PKG) (W.J. Thompson et al., 2000).

Active PKG inhibitsWnt/β-catenin signaling, on the onehand, by de-creasing β-cateninmRNA expression, and on the other hand, by depriv-ing β-catenin of the TCF/LEF complex, attaching it to active FOXO4

ulus, the transcriptional coactivator, β-catenin, is degraded by a multiprotein “destructioner/Thr kinases GSK3β and CK1, protein phosphatase 2A (PP2A), and the E3-ubiquitin ligase(LDL)-receptor related proteins 5 and 6), disheveled (Dvl) is activated. This inhibits the β-gen synthetase kinase 3b (GSK3β) (Moon et al., 2004), by activating PKB (Protein kinasen kinases, like activated PKG (Protein kinase G), also interfere with the β-catenin pathway.ansactivation activity, in combination with TCF, by sequestration of β-catenin by FOXO4.ors provokes the release of Gβ/γ subunits, which stimulate PKB through PI3K, and Gαs

ding of Gαs to axin releases GSK3β from the complex and GSK3β is phosphorylated andlso activates adenylate cyclase (AC) leading to the formation of cAMP and activation ofwith TCF/LEF, to the promoter region of several genes that are involved in proliferation,ctivity ofβ-catenin/TCF/LEF complex can be inhibitedby several other transcription factors,ber of NSAIDs (highlighted in red) suppress Wnt signaling by targeting several regulatorycoli protein; cAMP, cyclic adenosinemonophosphate; GSK3β; CK1α, casein kinaseα; COX,o; GSK3β, glycogen synthase kinase 3β; HDAC, histone deacetylase; LRP5/6, low densityK-1,3-phosphoinositide dependent protein kinase-1; PI3K, phosphatidylinositol 3-kinase;or-activated receptor gamma; TCF/Lef, T-cell factor/lymphoid enhancer factor; LEF, RXR,

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(Kwon et al., 2010; Li et al., 2013). In ApcMin mice, dietary sulindaceliminated, in particular, intestinal stem cells containing nuclear orphosphorylated β-catenin, indicating that targeting the Wnt/β-cateninpathway is a major mechanism to explain the cancer-preventing effectsof this drug (Qiu et al., 2010).

7.3. Interference of protein phosphatase2A with the β-catenin signaling pathway

Aspirin, diclofenac, sulindac sulfide (the pharmacologically activeme-tabolite of sulindac), and indomethacin diminished Wnt signaling at thelevel of gene transcription in different human colon cancer cell lines, irre-spective of COX-2 expression (Smith et al., 2000; Dihlmann et al., 2003;Gardner et al., 2004). This effect was apparently related to a decrease innuclear β-catenin localization and enhanced membranous β-cateninstaining in the human colon cancer cell line, SW480. In another study,Dihlmann et al. showed an increase in phosphorylated β-catenin proteincaused by aspirin that was not only due to enhanced GSK3β activity, butpossibly may have been triggered by inhibition of the specific serine/threonine-phosphatase, PP2A (Dihlmann et al., 2003; Bos et al., 2006),which is also a component of the β-catenin multiprotein “destructioncomplex” (Stamos & Weis, 2013). The phosphorylation status of β-catenin is important for its interaction with various binding partnersand also plays a pivotal role in the recruitment of co-activators to the nu-clear β-catenin/TCF/LEF complex (Daugherty & Gottardi, 2007). PP2A hasbeen shown to control the binding of CBP/p300 to β-catenin/TCF regulat-ed genes, leading to themaintenance of embryonic stem cell pluripotencyin mice (Miyabayashi et al., 2007). These data indicate that themolecularmechanisms of several NSAIDs in modifying Wnt/β-catenin signalingseem to be dependent on the cell type. Thus, further experiments areneeded to shed light on the direct molecular targets of these NSAIDswhich may relate to their anti-carcinogenic effects and their interferencewith the Wnt signaling pathway.

7.4. Interference of other transcription factors with Wnt signaling pathway

SomeNSAIDs, like diclofenac, aspirin, sulindac (and its metabolites),ibuprofen, and celecoxib, have been shown to exert their anti-carcinogenic effects through manipulation of the NF-κB signaling(Grösch et al., 2006; Gurpinar et al., 2013). Diclofenac and ibuprofenactivate the NF-κB pathway, leading to enhanced nuclear expressionof p65, which forms a complex with β-catenin, thereby, inhibitingthe transcription of β-catenin-dependent genes (Cho et al., 2005;Greenspan et al., 2011). Whether this mechanism also extends toother NSAIDS remains unclear and needs further investigation.

Besides NF-κB, the transcription factor-complex, PPARγ/RXRα (Per-oxisome Proliferator-Activated Receptor γ/Retinoid X receptor α), hasbeen shown to be a physical interaction partner of β-catenin, leadingto repression of β-catenin-dependent transcription (Lu et al., 2005).Eighteen of nineteen tested NSAIDs inhibit β-catenin-dependent tran-scription by interferencewith PPARγ/RXRα (Lu et al., 2005). Themolec-ular mechanism is not clear, but transactivation between PPARγ/RXRαand β-catenin seems to be important, because neither PPARγ norRXRα alone nor a mutant PPARγ protein, lacking transactivation activi-ty, account for the inhibitory effect of NSAIDs on β-catenin-dependenttranscription. For celecoxib, sulindac sulfide, indomethacin and others,a direct interaction with PPARγ or RXRα has been shown. Besides NF-κB, the transcription factor complex, PPARγ/RXRα, has been found tobe a physical interaction partner of β-catenin, leading to repression ofβ-catenin-dependent transcription (Lu et al., 2005).

7.5. Interference of 3-phosphoinositide-dependent protein kinase-1/PDK1/Akt signaling pathwaywith theWnt/β-catenin signaling pathway

Fig. 3 indicates that cytosolicβ-catenin stabilization/degradation canbe regulated by PI3K, PDK1, and protein kinase B (PKB/Akt) in an EP2/4

dependent manner. However, the PI3K signaling pathway is also acti-vated by several other membrane receptors, like integrins, receptor ty-rosine kinases (such as EGFR, VEGFR, Her2neu), cytokine receptors (e.g.IL2R), or G-protein coupled receptors, such as the EP2 receptor (for anoverview see Hemmings & Restuccia, 2012). All of these receptorshave been shown to be involved in cancer development and are oftenderegulated in different cancer types, leading to constitutive activationof the PI3K→PKB/Akt signaling pathway and cancer progression(Vivanco & Sawyers, 2002; Hennessy et al., 2005). Several NSAIDshave been shown to target PDK1 or PKB/Akt directly, leading toinhibition of this and the downstream signaling pathway and to anti-carcinogenic effects in vitro and in vivo in mice (Arico et al., 2002;Baoping et al., 2004; Kulp et al., 2004; Lee et al., 2005; Fan et al., 2006;Setia et al., 2014). This might also be a major mechanism by whichcelecoxib interferes with the Wnt/β-catenin pathway, leading toreduced β-catenin expression and transcriptional activity in humancancer cells, irrespective of COX-2 expression and activity (Maieret al., 2005; Sareddy et al., 2013).

In summary, NSAIDs potently inhibit Wnt signaling by differentmodes of action, which contribute to the anti-carcinogenic effects ofthese substances in various cancer types. This is of particular relevancefor therapy of adenomas and adenocarcinomas in FAP patients andother tumors displaying elevated Wnt signaling due to mutations inpathway-regulatory proteins. Several studies have convincingly indicateda concomitant contribution of β-catenin and PGE2 signaling pathways tothe development of CSC (Ishimoto et al., 2010; Al-Kharusi et al., 2013;Femia et al., 2013) as well as a superior therapeutic efficacy against CRC,particularly for those NSAIDs inhibiting both pathways by COX-dependent as well as COX-independent mechanisms (Qiu et al., 2010).

7.6. Non-steroidal anti-inflammatory drugsaffecting Wnt signaling by inhibition of 5-lipoxygenase

As mentioned above, a number of NSAIDs including indomethacin,the COX-2 inhibitor celecoxib, and the active metabolite of sulindac,sulindac sulfide, have been shown to inhibit Wnt signaling (Maieret al., 2005).

Sulindac, indomethacin, or celecoxib are able to inhibit Wnt signal-ing, but only at high concentrations, exceeding those for the suppres-sion of COX-1/-2. Sulindac sulfide is able to inhibit the 5-LO enzymedirectly at clinically relevant concentrations (Steinbrink et al., 2010).Celecoxib also has been reported to suppress the synthesis of 5-LOproducts by direct interference with the 5-LO enzyme activity (Maieret al., 2008).

Recently, wewere able to show that the non-selective COX inhibitor,indomethacin, is capable of suppressing 5-LO product formation inmonocytic THP-1 cells at somewhat higher concentrations than thoseneeded for COX inhibition. It is worth noting that for inhibitorsmentioned above, including indomethacin and sulindac sulfide, the 5-LO-inhibitory concentrations were similar to those required forsuppression of Wnt signaling and the aberrant self-renewal of LSCs(Roos et al., 2014).

This led us to hypothesize that NSAIDs and other 5-LO inhibitorsmay suppressWnt signaling by targeting the 5-LO enzyme, as describedin Section 9.

To better understand the complex interaction between 5-LO inhibi-tors, the 5-LO enzyme, and components of the Wnt signal transductionmachinery we briefly introduce the 5-LO pathway here.

8. Introduction to the pro-inflammatoryenzyme 5-lipoxygenase and its inhibitors

8.1. The 5-lipoxygenase biosynthetic pathway

The enzyme 5-LO (Fig. 4A) is expressed in a tissue- and celldifferentiation-specific manner, primarily in leukocytes including

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neutrophils, basophils, eosinophils, monocytes/macrophages, dendriticcells, mast cells, and B-lymphocytes (Radmark et al., 2007). The expres-sion level of 5-LO is regulated by promoter methylation and differentia-tion inducers [dimethyl sulfoxide (DMSO), retinoic acid, 1α, 2,5-dihydroxyvitamin D3 and TGF-β] upregulate 5-LO in immature myeloidcells (Radmark et al., 2007). Mechanistically, 5-LO catalyzes the inser-tion of molecular oxygen into polyunsaturated fatty acids, such as ara-chidonic acid (AA). Upon activation by Ca2+ and/or phosphorylationevents, 5-LO translocates from the cytosol or the nucleoplasm to the nu-clear envelope and (I) catalyzes the insertion ofmolecular oxygen at C-5

Fig. 4. Regulation of cellular 5-LO and COX activity. (A) Depending on the cell type, 5-LO reside2002). Though nuclear localization sequences are present in the primary sequence of 5-LO, thefactors such as testosterone (Pergola et al., 2008) or cell adherence (Brock, 2005) may affect tperinuclear regionwhere 5-LO product synthesis may occur (Werz, 2002; Brock, 2005; Newcomonists like platelet-activating factor (PAF), LTB4, formyl-methionyl-leucyl-phenylalanine (fMLP)translocates to the nuclear membrane (Werz, 2002) where it is anchored to phosphatidylcholiphorylation of 5-LO at the C-terminal catalytic domain can modulate its subcellular localizationprotein kinase (MK)-2/3 (Werz et al., 2000) and ERK1/2 (Werz et al., 2002a, 2002b) phosphorunsaturated fatty acids (i.e. AA) (Werz et al., 2002b). Interestingly, phosphorylation of 5-LO isWerz et al., 2001; Fredman et al., 2014) where it is anchored to phosphatidylcholine (PC) in aof 5-LO at the C-terminal catalytic domain can modulate its subcellular localization as well as5-LO (at Ser523), thus impairing the catalytic activity of 5-LO (Luo et al., 2004). Moreover, mand monocytes (Pergola et al., 2008; Pergola et al., 2011). Novel phosphorylation sites were renases Fgr, hematopoietic cell kinase (HCK) and Yes (Markoutsa et al., 2014). In parallel to 5-LOand releases AA from phospholipids (Leslie, 2004). The free AA is then transferred by the 5-LO-tabolism to LTA4 (Evans et al., 2008). FLAP is essential for cellular 5-LO product synthesis, and tfunction as a scaffold for 5-LO at the nuclear membrane (Mandal et al., 2008; Bair et al., 2012). Isequent transformation of LTA4 to LTB4 and LTC4, respectively (Newcomer & Gilbert, 2010). (B)membrane and releases AA from phospholipids. Two isoforms of cyclooxygenase, COX-1 and COther converted by a number of synthases to various eicosanoids, including PGE2, PGI2, and TXA2

different physiological processes including gastrointestinal integrity. COX-2 is detected negligibsites of inflammation. AA, arachidonic acid, CLP, coactosine-like protein; cPLA2, cytosolic phospkinase; LT, leukotriene;MK-2/3,MAPK-activatedprotein kinase; PKA, protein kinase A; PGE2, prtaglandin E2, TXA2 Thromboxane A2, TXAS, Thromboxane A2 synthase.

of AA that is provided by the5-LO-activating protein (FLAP), yielding theintermediate 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoicacid (5(S)-HpETE). Furthermore, it catalyzes (II) the dehydration of5(S)-HpETE to the epoxide leukotriene (LT)A4 (Radmark et al., 2015).Alternatively, 5(S)-HpETE can be reduced by glutathione peroxidases(GPx) to the corresponding alcohol, 5(S)-HETE,which can be further ox-idized to 5-oxo-ETE (Powell & Rokach, 2015).

Dependingon the cell type and enzymes expressed, LTA4 can be con-verted by soluble LTA4 hydrolase to LTB4 and/or conjugated with gluta-thione by LTC4 synthase to yield LTC4 (Bair et al., 2012).

s as a soluble protein either in the cytosol or in the nucleoplasm of resting cells (O. Werz,way in which the distinct subcellular localization is organized is unknown, but systemic

he subcellular redistribution. In activated cells, however, 5-LO is consistently found at theer & Gilbert, 2010; Bair et al., 2012). Upon cell activation by Ca2+-ionophores, soluble ag-and C5a, aswell as phagocytic particles such as zymosan, urate or phosphate crystals, 5-LOne (PC) in a Ca2+-dependent manner via the C2-like domain (Kulkarni et al., 2002). Phos-as well as its activation for product synthesis. The p38 MAPK-regulated MAPK-activatedylate 5-LO at Ser-271 and Ser-663, respectively, an action which is strongly promoted byassociated with increased accumulation of 5-LO at the nuclear membrane (Werz, 2002;Ca2+-dependent manner via the C2-like domain (Kulkarni et al., 2002). Phosphorylationits activation for product synthesis. Protein kinase A was also reported to phosphorylateoderate stimulation of ERK by androgens reduced 5-LO product formation in neutrophilscently identified at Tyr42, Tyr53 and either Tyr94 or Tyr445 phosphorylated by the Src ki-, the Ca2+-dependent cytosolic phospholipase A2 (cPLA2) moves to the nuclear membraneactivating protein (FLAP), an integral nuclear membrane protein, to 5-LO for efficient me-ogether with coactosine-like protein (CLP) that interacts with 5-LO as well, FLAP seems ton addition, LTC4S and LTA4Hmay participate in this multi-protein assembly to enable sub-Cyclooxygenase pathway. Cytosolic phospholipase A2 (cPLA2) translocates to the nuclearX-2, catalyze the conversion of arachidonic acid to prostaglandin H2 (PGH2), which is fur-

. COX-1 is constitutively expressed inmany tissues and is important for themaintenance ofly inmost tissues but can be rapidly and strongly induced by cytokines and cellular stress atholipase A2, FLAP, 5-LO activating protein; COX, cyclooxygenase; HCK, hematopoietic cellostaglandin E2, PGES, prostaglandin E2 synthase, PGIS, prostaglandin I2 synthase, PGI2 pros-

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Release of LTC4 into the extracellular environment and successiveamino acid (γ-glutamyl residue and glycine) cleavage yields LTD4 andthen LTE4. LTB4 is a potent chemotactic and chemokinetic mediator, ac-tivates granulocytes for degranulation, superoxide and nitrogen oxideproduction, enhances the phagocytic activity of neutrophils andmacro-phages, and stimulates secretion of immunoglobulins by lymphocytes(Haeggstrom & Funk, 2011). The cys-LTs, in particular LTD4, causesmooth muscle contraction, mucus secretion, vasoconstriction, plasmaextravasation, recruitment of eosinophils, and fibrocyte proliferation(Peters-Golden & Henderson, 2007; Haeggstrom & Funk, 2011).

Thus, LTs play roles in the immune response and in the pathogenesisof inflammatory diseases, such as arthritis, asthma, allergic rhinitis, andother chronic inflammatory disorders (Peters-Golden & Henderson,2007; Haeggstrom & Funk, 2011). Furthermore, 5-LO products contrib-ute to atherosclerosis (Poeckel & Funk, 2010) andmay regulate prolifer-ation and survival of cancer cells (Greene et al., 2011). Finally, 5-oxo-ETE may play important roles in asthma and allergic diseases, cancer,and cardiovascular disease as well (Powell & Rokach, 2015). The pro-inflammatory and immunomodulatory actions of LTs are mediatedby specific G protein-coupled receptors (GPCRs), the BLT1 and BLT2receptors for LTB4 and the CysLT1 and CysLT2 receptors for cys-LTs(Back et al., 2011).

8.2. 5-LO inhibitors

In order to intervene pharmacologically with 5-LO product synthe-sis, inhibition of cPLA2, antagonism of FLAP, and inhibition of 5-LO areall conceivable strategies (see (Pergola & Werz, 2010) for review). Di-rect 5-LO inhibitors can be categorized into four classes, according totheir inhibitorymode of action: (1) redox-active 5-LO inhibitors that re-duce the active site iron or scavenge activating hydroperoxides andthereby uncouple the catalytic cycle of the 5-LO enzyme; (2) iron-chelating agents, containing hydroxamic acid or N-hydroxyurea groupsthat form complexes with the active site iron; (3) non-redox-type in-hibitors that compete with AA or lipid hydroperoxides for binding to5-LOdevoid of redox properties; and (4) allosteric inhibitors that essen-tially act at the regulatory C2-like domain, e.g., by antagonizing thestimulatory effect of ATP or by interference with stimulatory effects ofphospholipids or diglycerides. In particular, for the latter group, severalstructurally different compounds with apparently distinct binding sites[e.g. indirubins at the ATP-binding site (Pergola et al., 2014), hyperforinat the PC-binding site (Feisst et al., 2009)] were recently reported withpromising molecular mechanisms and favorable pharmacodynamics.Nevertheless, despite intensive efforts to develop 5-LO inhibitors andthe resulting plethora of reported compounds, only the iron-ligand che-lator, zileuton, has become amarketed drug. In fact, all other 5-LO inhib-itors so far investigated failed in preclinical or clinical studies due toside-effects (e.g. methemoglobin formation, liver toxicity), pharmacoki-netic issues, or lack of efficacy (Pergola & Werz, 2010). Especially withnon-redox-type 5-LO inhibitors, phosphorylation of 5-LO and an elevat-ed oxidative tone in the cell can decrease their potency (O. Werz &Steinhilber, 2005).

9. Modulation by 5-LO and 5-LOinhibitors of Wnt signaling in leukemic stem cells

Over the last couple of years, evidence has been increasing for a keyrole of deregulated canonicalWnt signaling in the pathogenesis of hema-tological malignancies (HMs), though the exact underlying mechanismsremain poorly understood (Seke Etet et al., 2013). In AML and CML, therole of Wnt-signal activation for the pathogenesis and the maintenanceof the LSCs is well recognized (Zhao et al., 2007; Wang et al., 2010;Heidel et al., 2012). Deregulated activation ofWnt signaling leads to aber-rant self-renewal of the LSC in AML and CML and is fundamental to LSCmaintenance (Zhao et al., 2000; Passegue et al., 2003; Jamieson et al.,2004). Furthermore, it has been shown that not only Wnt signaling, but

also 5-LO is important for the maintenance of LSCs and the pathogenesisof AML and CML (Y. Chen et al., 2009; Roos et al., 2014). Although only asmall number of publications have addressed the role of 5-LO in the path-ogenesis of HMs, there is increasing evidence for an important role of 5-LO in the development of these diseases.

9.1. Disturbed 5-LO expression in leukemic cells

Differential 5-LO expression has been found in several types of pri-mary leukemic cells from patients with pre-B-cell ALL (Feltenmarket al., 1995; Vincent et al., 2008), CML (Radich et al., 2006; Grahamet al., 2007), AML (Valk et al., 2004; Vincent et al., 2008), B-cell chroniclymphocytic leukemia (B-CLL) (Rosenwald et al., 2001; Stratowa et al.,2001; Runarsson et al., 2005; Guriec et al., 2014), and mantle cell lym-phoma (MCL) (Boyd et al., 2009; Mahshid et al., 2009). Intriguingly,all these types of HM exhibit a deregulated Wnt/β-catenin signaling(Roman-Gomez et al., 2007; Zhao et al., 2007; Gelebart et al., 2008;Wu et al., 2009; Chung et al., 2010; Trowbridge et al., 2010; Wanget al., 2010; Kuhnl et al., 2011; Perez-Galan et al., 2011; Tandon et al.,2011; Heidel et al., 2012), suggesting an interrelation between 5-LOfunction and the Wnt/β-catenin signaling pathway.

One of the first studies to indicate involvement of 5-LO in HMs wasperformedby Tskuada et al. in 1986. In this study, treatment of leukemiccells with the competitive 5-LO inhibitor, AA-861, led to reduced cellproliferation which was accompanied by a reduction in cellular DNA,RNA, and protein synthesis. One of the conclusions of this study wasthat LTs play an important role in cancer cell viability (Tsukada et al.,1986). Similar results were obtained by Anderson et al., who observedthat primary human CML blast cells in culture, as well as leukemic celllines, showed reduced cell proliferation after treatment with different5-LO inhibitors (Anderson et al., 1995). Additionally, 5-LO inhibitionby AA-861 increased apoptosis in eicosapentaenoic acid (EPA)-treatedleukemic HL-60 cells (Gillis et al., 2007).

In a subsequent study, inhibition of 5-LO and FLAP was found to in-duce apoptosis inMCL cell lines and primary CLL cells, suggesting an im-portant role for 5-LO and/or its products in MCL and other B cellmalignancies. This was supported by the fact that 5-LO was up-regulated ~7-fold in MCL cells compared to normal B cells (Boyd et al.,2009). In addition, Runarsson et al. observed that treatment of B-CLLcells with the iron-ligand 5-LO inhibitor BWA4C and FLAP inhibitorMK886 counteracted CD40-dependent activation of these cells byinhibiting CD40-induced DNA synthesis and CD40-induced expressionof CD23, CD54, and CD150. This finding was likely LT-dependent, sinceaddition of exogenous LTB4 almost completely reversed the observedeffects (Runarsson et al., 2005).

It was also shown that the highly progressing disease in B-CLL pa-tients is associated with overexpression of 5-LO and other componentsof the 5-LO pathway, such as cPLA2 and BLT2 (Guriec et al., 2014). Theexpression levels of BLT1 and LTA4 hydroxylase were also significantlyraised in B-CLL cells compared to healthy B-cells. However, no statisti-cally significant correlation was found with the stage of the disease.Increased cPLA2α and LTC4 synthase expression was also found inleukemic granulocytes from AML patients. However, the formation ofLTB4 was also suppressed in the same patient samples (Stenke et al.,1998; Runarsson et al., 2007).

Taken together, it has been shown that in many different types ofleukemia there is an altered expression level of 5-LO protein and otherprotein components involved in LT biosynthesis suggesting a role ofthe 5-LO pathway in the pathogenesis of leukemia. Additionally, inhib-itor studies revealed a function of 5-LO products in proliferation andapoptosis of leukemic cells with particular relevance to B-CLL cells.

9.2. 5-LO and acute myeloid leukemia

Recently, it was shown that 5-LO is strongly upregulated in AML1/ETO-positive AML (DeKelver et al., 2013). The fusion protein AML1/

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ETO is the result of the chromosomal t(8;21) translocation (transfer ofgenetic material between chromosome 8 and chromosome 21), whichtogether with t(15;17) (PML/RARα) is one of the most common trans-locations associated with AML. These leukemia-associated fusion pro-teins (LAFP) are also known to induce aberrant activation of canonicalWnt signaling which, as a result, leads to an increased self-renewal ca-pacity of LSCs (Tenen, 2003; Muller-Tidow et al., 2004; Zheng et al.,2004; Zhao et al., 2007;Wang et al., 2010). DeKelver et al. analyzed pub-lically available microarray data from AML M2 patients with and with-out the t(8;21) translocation (Valk et al., 2004). They found that 5-LOexpression was ~2.3-fold higher in t(8;21) patients than in patientswithout the translocation. Notably, both patient groups had elevated5-LO levels relative to normal CD34+ control samples (DeKelver et al.,2013). Additionally, it could be shown, by gene expression microarrayand chip analysis, that 5-LO is a potential disease-related target geneof AML1/ETO (Lo et al., 2012). AML1/ETO, as well as its C-terminallytruncated leukemic isoform, AML1/ETOex9, is able to up-regulate 5-LOvia the transcription factor, KLF6 (Krppl-like factor 6), a protein whichis also known to be a cell-specific positive regulator of LTC4 synthase(Zhao et al., 2000). Furthermore, studies with KLF6−/− mice revealedthat 5-LO is also critically required for embryonic stem cell differentia-tion and early hematopoiesis (Matsumoto et al., 2006).

Thus, 5-LO is a direct disease-related and pathophysiologicallyrelevant target gene upregulated by the oncogenic leukemia associatedfusion protein, AML1/ETO, which is crucially involved in the inductionand maintenance of AML.

9.3. 5-LO and self-renewal capacity of leukemic cells

To analyze the effect of 5-LO on the in vitro self-renewal capacityof the LAFPs AML1/ETOex9, PML/RARα, and MLL-AF9 (translocationt(9;11)), DeKelver et al. performed serial replating assays using wildtype and 5-LO−/− murine total bone marrow cells that were trans-duced with either control vector or LAFPs. 5-LO deficiency led to de-creased in-vitro self-renewal capacity with all LAFPs. Remarkably, lossof 5-LO abolished completely the aberrant replating capacity of AML1/ETOex9-positive cells. Nevertheless, AML1/ETOex9-expressing 5-LO−/− cells were still capable of leukemia induction. These results ledthe authors to hypothesize that in the murine AML1/ETOex9 leukemiainduction model, other factors may exist that allow AML1/ETOex9to overcome these defects. It is also possible that 5-LO expressionmay be required to a greater extent by AML1/ETO than by AML1/ETOex9 cells in leukemia development, especially since AML1/ETO up-regulates KLF6 more strongly than AML1/ETOex9. Another reasoncould be that in the in-vitro and in-vivo cell culture experiments, theauthors used different cell populations, containing different ratios ofHSCs (total bonemarrow versus fetal liver cells), suggesting that differ-ent subtypes of HSCs respond differently to the introduction of AML1/ETOex9 and 5-LO deficiency (DeKelver et al., 2013).

Taken together, these experiments demonstrated the impact of 5-LOon the self-renewal capacity of LSC. Thus, 5-LO seems to be involved inthe maintenance of the undifferentiated state of these leukemic cellswhich is a characteristic feature of LSCs.

9.4. 5-LO and human leukemic stem cell function

5-LO also seems to be involved in human CML stem cell function, asrevealed by microarray studies. In these studies, 5-LO was differentiallyexpressed in human CD34+ CML cells, compared to CD34+ controlcells (Radich et al., 2006; Graham et al., 2007). In a recent publication,the expression levels of 5-LO and BLT1 were decreased both in bloodsamples and CD34+ stem cells from t(9;22)-positive CML patients har-boring the BCR/ABL fusion protein, compared to samples from healthydonors (Lucas et al., 2014). On the other hand, it was shown that 5-LOis up-regulated in BCR/ABL-positive GFP+Lin−ckit+Sca1+ LSCs in com-parison to non-BCR/ABL expressing GFP+Lin−ckit+Sca1+ cells and that

5-LO is indispensable for the aberrant self-renewal capacity of theseLSCs and the development of CML in mice (Y. Chen et al., 2009). Thiswas revealed using amurine BCR/ABL leukemia inductionmodel. Recip-ients of BCR/ABL transduced bone marrow cells from 5-LO−/− donormice failed to develop CML, whereas recipients of BCR/ABL transducedbone marrow cells from wild type donor mice developed the diseaseand died within 4 weeks. In the absence of 5-LO, myeloid leukemiacells gradually disappeared in the CML mice. A functional defect in nor-mal HSCs could not be detected. 5-LO deficiency led only to an impair-ment of LT-LSCs, preventing these cells from developing CML. Thisdefective function of LSCs in the absence of 5-LO was correlated with areduction ofβ-catenin expression levels, suggesting a further regulatorylink between 5-LO and Wnt signaling.

The effect of 5-LO deficiency on BCR/ABL-positive LSCs was furtherinvestigated in studies using the approved iron-chelating 5-LO inhibitorzileuton. Zileuton led to impairment of BCR/ABL positive LSCs in a sim-ilar manner to that observed with 5-LO knock-out. Pharmacologicalsuppression of 5-LO by zileuton in mice with BCR/ABL-induced CMLprolonged their survival compared to control mice. An even better ther-apeutic effect could be achieved with imatinib, a first generation BCR/ABL tyrosine kinase inhibitor, in combination with zileuton. However,BCR/ABL-induced lymphoid leukemia, which originates from morecommitted lymphoid progenitors than BCR/ABL-induced CML, was notaffected by 5-LO deficiency (S. Li et al., 1999), suggesting that 5-LOis specifically required by CML stem cells and not by ALL stem cells (Y.Chen et al., 2009). These results were followed by a clinical phase Istudy in which zileuton was tested in combination with imatinib forthe treatment of CML (Safety of Zileuton (Zyflo) in CombinationWith I-matinib Mesylate (Gleevec) in CML; http://clinicaltrials.gov trial No.NCT01130688). This study has been completed but the results still re-main to be published. Another phase I study for treatment of BCR/ABL-positive CMLwith zileuton in combinationwith dasatinib, a secondgeneration BCR/ABL tyrosine kinase inhibitor, is currently ongoing(Evaluating the Safety of Zileuton (Zyflo®) in Combination WithDasatinib (Sprycel®) in Chronic Myelogenous Leukemia; http://clinicaltrials.gov trial No. NCT02047149).

Recently, we demonstrated that 5-LO is also crucial for the mainte-nance of LSC in a PML/RARα-positive stem cell model of AML (Rooset al., 2014). First, we performed inhibitor studies on Sca1+Lin−murine hematopoietic stem and progenitor cells (HSPC). The HSPCwere either transducedwith PML/RARα or an empty vector. As 5-LO in-hibitors, we used the non-redox type inhibitor CJ-13,610 and zileuton.These inhibitors were both able to abolish the aberrant replating effi-ciency of PML/RARα-expressing HSPCs in a concentration dependentmanner. The same inhibitory effect could be observed on the LT- andST-stem cell capacity. No cytotoxic effect could be observed after treat-ment of the non-PML/RARα-positive control cells, which is consistentwith the work of Y. Chen et al. (2009). Furthermore, the occurrence ofstem cell suppression coincided with inhibition of Wnt signaling. Thiswas shownby immunohistochemical staining ofmurine spleen coloniesinduced by PML/RARα against β-catenin. Treatment with CJ-13,610 ledto reversal of the PML/RARα-activation of Wnt signaling.

Collectively, these experiments confirmadirect link between the 5-LOandWnt signaling pathways which is indispensable for the maintenanceof LSCs. Accordingly, genetic andpharmacological inhibition of 5-LO led toan impairment of the LSC in AML andCML cells supporting the hypothesisof an regulatory interaction of both pathways. Additionally, pharmacolog-ical inhibition of 5-LO significantly increased the therapeutic effect of ima-tinib, a classical therapeutic agent used in the standard therapy of CML.Thus, a combination of standard therapeutics and 5-LO inhibitors couldbe beneficial for the clinical outcome of these patients.

9.5. Potential mechanism of 5-LO interaction with Wnt signaling

To confirm and investigate more extensively the involvement of 5-LO in the regulation of the Wnt signaling pathway, we performed co-

57J. Roos et al. / Pharmacology & Therapeutics 157 (2016) 43–64

immunoprecipitation experiments as well as reporter gene assays andimmunofluorescence microscopy in different cancer cell lines. In thesestudies, we demonstrated that enzymatically inactive 5-LO traps β-catenin at the nuclear envelope (Fig. 5), prevents the translocation ofβ-catenin to the nucleus, and therefore, suppresses the PML/RARα-induced activation of Wnt target genes. Additionally, we observed that5-LO directly interacts with β-catenin (Roos et al., 2014), suggesting aconnection between Wnt signaling and the 5-LO pathway representedby a 5-LO/β-catenin association. However, an interaction of 5-LO withadditional components of the Wnt signaling pathway could not beexcluded. Furthermore, the exact molecular mechanism(s) by whichinactive 5-LO inhibits the translocation of β-catenin to the nucleus andinteracts with β-catenin and possibly other Wnt pathway componentsstill needs detailed experimental investigation.

9.6. 5-LO and Wnt signaling in solid tumors

Increasing evidence in the literature implicates 5-LO as a player inthe growth of several solid tumor types, including pancreatic, colorectal,prostate and breast cancers (Kort et al., 1992; Ding et al., 1999a, 1999b;Gupta et al., 2001; Hennig et al., 2002; Jiang et al., 2003; Soumaoro et al.,2006; Ihara et al., 2007; Bishayee & Khuda-Bukhsh, 2013) and the Wntsignaling pathway contributes to the pathogenesis of these cancers (Liuet al., 2000; Milovanovic et al., 2004; Yardy & Brewster, 2005;Yokoyama et al., 2014). However, even though a correlation between5-LO and Wnt signaling in solid tumors is obvious, at the moment nostudy has yet been published that directly addresses the relation be-tween 5-LO or the effects of 5-LO inhibitors and theWnt signaling path-way in these tumors types.

Fig. 5. Proposedmechanism ofWnt signaling suppression by 5-LO. Binding to β-catenin of enzysignaling pathway by preventing the translocation of β-catenin to the nucleus and subsequent gdeacetylase; TCF/LEF, T-cell factor/lymphoid enhancer factor.

10. Conclusion and future direction

Dysfunctional Wnt signaling is emerging as having pathophysiolog-ical significance in a number of diseases, including cancer, classicalinflammatory diseases, and neurodegenerative diseases triggered byneuroinflammatory processes.

Numerous high-throughput screens have been employed to obtainsmall-molecule targeted drugs that would interfere with componentsof Wnt signaling, mainly directed towards disruption of the binding ofβ-catenin to its nuclear target, TCF/LEF.

Many compounds with promising activity in cell culture have beenidentified and characterized, but translation into clinically applicabledrugs is still an apparently insurmountable obstacle due to lack ofselectivity and a number of pleiotropic off-target effects. This relatesparticularly to drugs that interfere with the proteasomal degradationof β-catenin, which results in severe side effects in normal tissue asnon-nuclearβ-catenin contributes to the stabilization of physiologicallyimportant intercellular adherens junctions.

The clinical value of silencingWnt signaling further upstream, i.e., atthe level of the Frizzled receptor, remains unclear as a number of tissuesdepend on a certain basic Wnt activity which may be associated withunknown adverse side effects.

Thus, a complex mixture of problems is encountered in developingWnt inhibitors using in silico methods or high-throughput screensusing compound libraries.

Several inhibitors of eicosanoid signaling, such as NSAIDs, have beenshown to interfere in a clinically relevant manner with Wnt signaling,by COX/PGE2 aswell as COX/PGE2-independentmechanisms and there-by, induce Wnt-dependent anti-tumorigenic effects (see Section 7).

matically inactive 5-LO, after treatmentwith the 5-LO inhibitor CJ-13610, inhibits theWntene transcriptional activity. For further details see Section 9. GRG, Groucho; HDAC, histone

58 J. Roos et al. / Pharmacology & Therapeutics 157 (2016) 43–64

Mechanistically, NSAIDs differ widely from classical Wnt inhibitors intheir actions on the Wnt signal transduction cascade and follow novelmechanistic routes of suppression ofWnt signaling. Furthermore, sever-al very recent studies presented here provide valid and comprehensiveevidence for an essential role of 5-LO in and interconnection with Wntsignaling in the pathogenesis of leukemia and the maintenance ofLSCs in certain sub-types of leukemia.

As a consequence, pharmacological 5-LO inhibition,with concomitantsuppression of Wnt signaling, is a potential novel approach to adjuvantstem cell therapy in AML and CML as well as other Wnt-dependent dis-eases, such as breast cancer, human head and neck squamous cell carci-nomas, and some inflammatory diseases (see Section 4) (Liu et al., 2013).

Further insight into the value of anti-5-LO therapy is likely to begained from two clinical trials — the recently finished study withzileuton and imatinib as combined treatment for CML, as well as thenew study with zileuton and dasatinib. Yet to fully understand therole of 5-LO in the Wnt signaling pathway (and possible otherleukemia-related pathways), more detailed and comprehensive studiesare necessary.

A number of approved NSAIDs, administered at high doses, suppressWnt signaling in patients but without causing severe concomitantsystemic toxicity in the organism. The majority of side effects ofthese drugs arewell documented and derives from suppression of pros-taglandin biosynthesis. This includes mainly cardiovascular and throm-boembolic events and gastrointestinal ulceration (Bhala et al., 2013).However, there are no reports of adverse effects or toxicity directly aris-ing from the Wnt-inhibitory activity of NSAIDs.

Thus, suppression of Wnt signaling by NSAIDs may follow advanta-geous mechanistic strategies and NSAIDs as well as 5-LO inhibitors,therefore, should be reassessed as a basis for the design of a new classof Wnt inhibitors.

Following the clarification of the exact molecular mechanism bywhich these drugs inhibitWnt signaling, it seems reasonable that chem-ical structures responsible for Wnt inhibition could be separated fromthe structural elements causing suppression of COX enzymes. The factthat a number of drugs targeting eicosanoid synthesis concomitantlysuppress Wnt signaling independently of COX could further indicate apossible common, but still unidentified key target for the regulation ofWnt activity.

In this respect, 5-LO inhibitors, such as CJ-13610, activate a poten-tially naturally occurring novel 5-LO-dependentmechanism of suppres-sion of Wnt signaling that is non-toxic, at least in mice, and might bewell tolerated in patients.

Future careful studies are needed to clarify the regulatory mecha-nism linking eicosanoid synthesis pathways to oncogenicWnt signalingas a potential basis for novel anti-inflammatory drugs that concomitant-ly suppress Wnt signaling.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.pharmthera.2015.11.001.

Conflict of interest statement

MJP is a consultant to Leo Pharma a/s and Xellia PharmaceuticalsApS. The other authors declare that there are no conflicts of interest.

Acknowledgment

Thisworkwas funded by theGermanResearch Foundation (DFGpro-jects MA-5825/1-1, GR2011/3-2) and the DFG SonderforschungsbereichSFB 1039/1, the Graduate School TRIP (Translational Research Innova-tion — Pharma), the European Research Council under the EuropeanUnion's Seventh Framework Programme (FP7/2007–2013; ERC grant268309) and the LOEWE Lipid Signaling Forschungszentrum Frankfurt(LIFF). MJP is supported by LOEWE Research Centre for TranslationalMedicine and Pharmacology, financed by the State of Hessen. T.J.M. is

recipient of a Heisenberg fellowship of the German Research Foundation(DFG-MA-5825-2-1).

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